PROTEIN VARIANTS HAVING MODIFIED IMMUNOGENICITY

- NOVOZYMES A/S

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein comprising the steps obtaining antibody binding peptide sequences, using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein, defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence, changing one or more of the amino acids defining the epitope area of the parent protein by genetical engineering mutations of a DNA sequence encoding the parent protein, introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and evaluating the immunogenicity of the protein variant using the parent protein as reference. The invention further relates to the protein variant and use thereof, as well as to a method for producing said protein variant.

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Description
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 09/957,806 filed on Sep. 21, 2001, which is a continuation of PCT/DK01/00293 filed Apr. 30, 2001 and claims, under 35 U.S.C. 119, priority or the benefit of Danish application nos. PA 2000 00707 and PA 2001 00327 filed Apr. 28, 2000 and Feb. 28, 2001, respectively, and U.S. application Nos. 60/203,345 and 60/277,817 filed May 10, 2000 and Mar. 21, 2001, respectively, the contents of which are fully incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein, to the protein variant and use thereof, as well as to a method for producing said protein variant.

BACKGROUND OF THE INVENTION

An increasing number of proteins, including enzymes, are being produced industrially, for use in various industries, housekeeping and medicine. Being proteins they are likely to stimulate an immunological response in man and animals, including an allergic response.

Depending on the application, individuals get sensitised to the respective allergens by inhalation, direct contact with skin and eyes, or injection. The general mechanism behind an allergic response is divided in a sensitisation phase and a symptomatic phase. The sensitisation phase involves a first exposure of an individual to an allergen. This event activates specific T- and B-lymphocytes, and leads to the production of allergen specific IgE antibodies (in the present context the antibodies are denoted as usual, i.e. immunoglobulin E IgE etc.). These IgE antibodies eventually facilitate allergen capturing and presentation to T-lymphocytes at the onset of the symptomatic phase. This phase is initiated by a second exposure to the same or a resembling antigen. The specific IgE antibodies bind to the specific IgE receptors on mast cells and basophils, among others, and capture at the same time the allergen. The polyclonal nature of this process results in bridging and clustering of the IgE receptors, and subsequently in the activation of mast cells and basophils. This activation triggers the release of various chemical mediators involved in the early as well as late phase reactions of the symptomatic phase of allergy. Prevention of allergy in susceptible individuals is therefore a research area of great importance.

For certain forms of IgE-mediated allergies, a therapy exists, which comprises repeated administration of allergen preparations called ‘allergen vaccines’ (Int. Arch. Allergy Immunol., 1999, vol. 119, pp 1-5). This leads to reduction of the allergic symptoms, possibly due to a redirection of the immune response away from the allergic (Th2) pathway and towards the immunoprotective (Th1) pathway (Int. Arch. Allergy Immunol., 1999, vol. 119, pp 1-5).

Various attempts to reduce the immunogenicity of polypeptides and proteins have been conducted. It has been found that small changes in an epitope may affect the binding to an antibody. This may result in a reduced importance of such an epitope, maybe converting it from a high affinity to a low affinity epitope, or maybe even result in epitope loss, i.e. that the epitope cannot sufficiently bind an antibody to elicit an immunogenic response.

There is a need for methods to identify epitopes on proteins and alter these epitopes in order to modify the immunogenicity of proteins in a targeted manner. Such methods and kits for their execution can have at least four useful purposes:

1) reduce the allergenicity of a commercial protein using protein engineering.

2) reduce the potential of commercial proteins to cross-react with environmental allergens and hence cause allergic reactions in people sensitized to the environmental allergens (or vice versa).

3) improve the immunotherapeutic effect of allergen vaccines.

4) assist characterization of clinical allergies in order to select the appropriate treatment, including allergen vaccination.

In WO 99/53038 (Genencor Int.) as well as in prior references (Kammerer et al, Clin. Exp. Allergy, 1997, vol. 27, pp 1016-1026; Sakakibara et al, J. Vet. Med. Sci., 1998; vol. 60, pp. 599-605), methods are described, which identify linear T-cell epitopes among a library of known peptide sequences, each representing part of the primary sequence of the protein of interest. Further, several similar techniques for localization of B-cell epitopes are disclosed by Walshet et al, J. Immunol. Methods, vol. 121, 1275-280, (1989), and by Schoofs et al. J. Immunol. vol. 140, 611-616, (1987). All of these methods, however, only leads to identification of linear epitopes, not to identification of ‘structural’ or ‘discontinuous’ epitopes, which are found on the 3-dimensional surface of protein molecules and which comprise amino acids from several discrete sites of the primary sequence of the protein. For several allergens, it has been realized that the dominant epitopes are of such discontinuous nature (Collins et al., Clin. Exp. All. 1996, vol. 26, pp. 36-42).

Slootstra et al; Molecular Diversity, 2, pp. 156-164, 1996 disclose the screening of a semi-random library of synthetic peptides for their binding properties to three monoclonal antibodies by immobilizing the peptides on polyethylene pins and binding a dilution series of each antibody to the pins. This reference does not disclose any indication of how the antibody binding peptide sequences relate to any full protein antigens or allergens.

In WO 92/10755 a method for modifying proteins to obtain less immunogenic variants is described. Randomly constructed protein variants, revealing a reduced binding of antibodies to the parent enzyme as compared to the parent enzyme itself, are selected for the measurement in animal models in terms of allergenicity. Finally, it is assessed whether reduction in immunogenicity is due to true elimination of an epitope or a reduction in affinity for antibodies. This method targets the identification of amino acids that may be part of structural epitopes by using a complete protein for assessing antigen binding. The major drawbacks of this approach are the ‘trial and error’ character, which makes it a lengthy and expensive process, and the lack of general information on the epitope patterns. Without this information, the results obtained for one protein can not be applied on another protein.

WO 99/47680 (ALK-ABELLÓ) discloses the identification and modification of B-cell epitopes by protein engineering. However, the method is based on crystal structures of Fab-antigen complexes, and B-cell epitopes are defined as “a section of the surface of the antigen comprising 15-25 amino acid residues, which are within a distance from the atoms of the antibody enabling direct interaction” (p. 3). This publication does not show how one selects which Fab fragment to use (e.g. to target the most dominant allergy epitopes) or how one selects the substitutions to be made. Further, their method cannot be used in the absence of such crystallographic data for antigen-antibody complexes, which are very cumbersome, sometimes impossible, to obtain—especially since one would need a separate crystal structure for each epitope to be changed.

Hence, it is of interest to establish a general and efficient method to identify structural epitopes on the 3-dimensional surface of commercial and environmental allergens.

SUMMARY OF THE INVENTION

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to a parent protein, comprising the steps of:

a) obtaining antibody binding peptide sequences,

b) using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein,

c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence,

d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein,

e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and

f) evaluating the immunogenicity of the protein variant using the parent protein as reference.

A second aspect of the present invention is a protein variant having modified immunogenicity as compared to its parent protein. The amino acid sequence of the protein variant differs from the amino acid sequence of the parent protein with respect to at least one epitope pattern of the parent protein, such that the immunogenicity of the protein variant is modified as compared with the immunogenicity of the parent protein.

A further aspect of the present invention is a composition comprising a protein variant as defined above, as well as the use of the composition for industrial application, such as the production of a formulation for personal care products (for example shampoo; soap; skin, hand and face lotions; skin, hand and face crèmes; hair dyes; toothpaste), food (for example in the baking industry), detergents and for the production of pharmaceuticals, e.g. vaccines.

Yet another aspect is a DNA molecule encoding a protein variant as defined above.

Further aspects are a vector comprising a DNA molecule as described above as well a host cell comprising said DNA molecule.

Another aspect is a method of producing a protein variant having modified immunogenicity as compared to the parent protein as defined above.

DEFINITIONS

Prior to a discussion of the detailed embodiments of the invention, a definition of specific terms related to the main aspects of the invention is provided.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”) DNA Cloning: A Practical Approach, Volumes I and II/D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984).

When applied to a protein, the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure. When applied to a polynucleotide molecule, the term “isolated” indicates that the molecule is removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, and may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985).

A “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary or quaternary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

A DNA “coding sequence” is a double-stranded DNA sequence, which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

An “Expression vector” is a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

A “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a “secretory peptide” that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term “promoter” is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.

“Operably linked”, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.

A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.

“Isolated polypeptide” is a polypeptide which is essentially free of other non-[enzyme]polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.

“Heterologous” DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell.

A cell has been “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. Preferably, the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

A “clone” is a population of cells derived from a single cell or common ancestor by mitosis.

“Homologous recombination” refers to the insertion of a foreign DNA sequence of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.

Nucleic Acid Sequence

The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain producing the polypeptide, or from another related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated” nucleic acid sequence as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electorphoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Nucleic Acid Construct

As used herein the term “nucleic acid construct” is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term “construct” is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The construct may optionally contain other nucleic acid segments.

The DNA of interest may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., supra).

The nucleic acid construct may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.

The nucleic acid construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term “coding sequence” as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention when placed under the control of the above mentioned control sequences. The boundaries of the coding sequence are generally determined by a translation start codon ATG at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term “control sequences” is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcription and translation control sequences which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the expressed polypeptide into the cell's secretory pathway of the host cell. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.

Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the secreted polypeptide. A foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion relative to the natural signal peptide coding region normally associated with the coding sequence. The signal peptide coding region may be obtained from a glucoamylase or an amylase gene from an Aspergillus species, a lipase or proteinase gene from a Rhizomucor species, the gene for the alpha-factor from Saccharomyces cerevisiae, an amylase or a protease gene from a Bacillus species, or the calf preprochymosin gene. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).

The nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.

An activator is a protein which activates transcription of a nucleic acid sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal 9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244; Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding an activator may be obtained from the genes encoding Bacillus stearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activator protein 1 (hap1), Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4), and Aspergillus nidulans ammonia regulation protein (areA). For further examples, see Verdier, 1990, supra and MacKenzie et al., 1993, Journal of General Microbiology 139:2295-2307.

A chaperone is a protein which assists another polypeptide in folding properly (Hartl et al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS 19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189; Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature 355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; Robinson et al., 1994, Bio/Technology 1:381-384). The nucleic acid sequence encoding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins, Aspergillus oryzae protein disulphide isomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae BiP/GRP78, and Saccharomyces cerevisiae Hsp70. For further examples, see Gething and Sambrook, 1992, supra, and Hartl et al., 1994, supra.

A processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlin and Ogrydziak, 1994, Yeast 10:67-79; Fuller et al., 1989, Proceedings of the National Academy of Sciences USA 86:1434-1438; Julius et al., 1984, Cell 37:1075-1089; Julius et al., 1983, Cell 32:839-852). The nucleic acid sequence encoding a processing protease may be obtained from the genes encoding Aspergillus niger Kex2, Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowia lipolytica dibasic processing endoprotease (xpr6).

It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be placed in tandem with the regulatory sequence.

Promoters

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis alkaline protease gene, the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80:21-25), or the Bacillus pumilus xylosidase gene, or by the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters. Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S. Pat. No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters. Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter.

Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4-c (Russell et al., Nature 304 (1983), 652-654) promoters.

Further useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

Examples of suitable promoters for directing the transcription of the DNA encoding the polypeptide of the invention in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814) or the adenovirus 2 major late promoter.

An example of a suitable promoter for use in insect cells is the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

Terminators

Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators.

Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

Polyadenylation Signals

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.

Polyadenylation sequences are well known in the art for mammalian host cells such as SV40 or the adenovirus 5 Elb region.

Signal Sequences

An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, the Rhizomucor miehei aspartic proteinase gene, the Humicola lanuginosa cellulase or lipase gene, or the Rhizomucor miehei lipase or protease gene, Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease. The signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral alpha-amylase, A. niger acid-stable amylase, or A. niger glucoamylase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

For secretion from yeast cells, the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell. The signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the a-factor signal peptide (cf. U.S. Pat. No. 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

For efficient secretion in yeast, a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the polypeptide. The function of the leader peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The leader peptide may be the yeast a-factor leader (the use of which is described in e.g. U.S. Pat. No. 4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.

For use in insect cells, the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. U.S. Pat. No. 5,023,328).

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, tetracycline, neomycin, hygromycin or methotrexate resistance. A frequently used mammalian marker is the dihydrofolate reductase gene (DHFR). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.

The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.

The vectors of the present invention may be integrated into the host cell genome when introduced into a host cell. For integration, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. These nucleic acid sequences may be any sequence that is homologous with a target sequence in the genome of the host cell, and, furthermore, may be non-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβ1. Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

More than one copy of a nucleic acid sequence encoding a polypeptide of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. The term “host cell” encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication.

The cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome. “Transformation” means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above.

The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. The transformation of a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278).

The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.

Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.

Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651), BHK (ATCC CRL 1632, 10314 and 1573, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987, Hawley-Nelson et al., Focus 15 (1993), 73; Ciccarone et al., Focus 15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). Representative groups of Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J. S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).

The yeast host cell may be selected from a cell of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, or Yarrowia. In a preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Other useful yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279 and U.S. Pat. No. 4,879,231).

In a preferred embodiment, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In a more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym thereof. In an even more preferred embodiment, the filamentous fungal host cell is an Aspergillus cell. In another even more preferred embodiment, the filamentous fungal host cell is an Acremonium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Fusarium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Humicola cell. In another even more preferred embodiment, the filamentous fungal host cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal host cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal host cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal host cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal host cell is a Trichoderma cell. In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger, Aspergillus nidulans or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium cell of the section Discolor (also known as the section Fusarium). For example, the filamentous fungal parent cell may be a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium reticulaturn, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, or Fusarium trichothecioides cell. In another preferred embodiment, the filamentous fungal parent cell is a Fusarium strain of the section Elegans, e.g., Fusarium oxysporum. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal host cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal host cell is a Myceliophthora thermophilum cell. In another most preferred embodiment, the filamentous fungal host cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal host cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris cell or an Acremonium chtysogenum cell. In another most preferred embodiment, the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.

Transformation

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147-156 or in copending U.S. Ser. No. 08/269,449. Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023. The transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156.

Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920. Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).

Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in U.S. Pat. No. 4,745,051; U.S. Pat. No. 4,775,624; U.S. Pat. No. 4,879,236; U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222; EP 397,485) all of which are incorporated herein by reference. The insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214). Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.

Methods of Production

The transformed or transfected host cells described above are cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media are prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).

If the molecules are secreted into the nutrient medium, they can be recovered directly from the medium. If they are not secreted, they can be recovered from cell lysates. The molecules are recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of molecule in question.

The molecules of interest may be detected using methods known in the art that are specific for the molecules. These detection methods may include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.

The molecules of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

The term “immunological response”, used in connection with the present invention, is the response of an organism to a compound, which involves the immune system according to any of the four standard reactions (Type I, II, III and IV according to Coombs & Gell).

Correspondingly, the “immunogenicity” of a compound used in connection with the present invention refers to the ability of this compound to induce an ‘immunological response’ in animals including man.

The term “allergic response”, used in connection with the present invention, is the response of an organism to a compound, which involves IgE mediated responses (Type I reaction according to Coombs & Gell). It is to be understood that sensibilization (i.e. development of compound-specific IgE antibodies) upon exposure to the compound is included in the definition of “allergic response”.

Correspondingly, the “allergenicity” of a compound used in connection with the present invention refers to the ability of this compound to induce an ‘allergic response’ in animals including man.

The term “parent protein” refer to the polypeptide to be modified by creating a library of diversified mutants. The “parent protein” may be a naturally occurring (or wild-type) polypeptide or it may be a variant thereof prepared by any suitable means. For instance, the “parent protein” may be a variant of a naturally occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.

The term “enzyme variants” or “protein variants” refer to a polypeptide of the invention comprising one or more substitutions of the specified amino acid residues. The total number of such substitutions is typically not more than 10, e.g. one, two, three, four, five or six of said substitutions. In addition, the enzyme variant or protein variant of the invention may optionally include other modifications of the parent enzyme, typically not more than 10, e.g. not more than 5 such modifications. The variant generally has a homology with the parent enzyme of at least 80%, e.g. at least 85%, typically at least 90% or at least 95%.

The term “randomized library” of protein variants refers to a library with at least partially randomized composition of the members, e.g. protein variants.

An “epitope” is a set of amino acids on a protein that are involved in an immunological response, such as antibody binding or T-cell activation. One particularly useful method of identifying epitopes involved in antibody binding is to screen a library of peptide-phage membrane protein fusions and selecting those that bind to relevant antigen-specific antibodies, sequencing the randomized part of the fusion gene, aligning the sequences involved in binding, defining consensus sequences based on these alignments, and mapping these consensus sequences on the surface or the sequence and/or structure of the antigen, to identify epitopes involved in antibody binding.

By the term “epitope pattern” is meant such a consensus sequence of antibody binding peptides. An example is the epitope pattern A R R<R. The sign “<” in this notation indicates that the aligned antibody binding peptides included a non-consensus amino acid between the second and the third arginine.

An “epitope area” is defined as the amino acids situated close to the epitope sequence amino acids. Preferably, the amino acids of an epitope area are located <5 Å from the epitope sequence. Hence, an epitope area also includes the corresponding epitope sequence itself. Modifications of amino acids of the ‘epitope area’ can possibly affect the immunogenic function of the corresponding epitope.

By the term “epitope sequence” is meant the amino acid residues of a parent protein, which have been identified to belong to an epitope by the methods of the present invention (an example of an epitope sequence is E271 Q12 I8 in Savinase).

The term ‘antibody binding peptide’ denotes a peptide that bind with sufficiently high affinity to antibodies. Identification of ‘antibody binding peptides’ and their sequences constitute the first step of the method of this invention.

“Anchor amino acids” are the individual amino acids of an epitope pattern.

“Hot spot amino acids” are amino acids of parent protein, which are particularly likely to result in modified immunogenecity if they are mutated. Amino acids, which appear in three or more epitope sequences or which correspond to anchor amino acids are hot spot amino acids.

“Environmental allergens” are protein allergens that are present naturally. They include pollen, dust mite allergens, pet allergens, food allergens, venoms, etc.

“Commercial allergens” are protein allergens that are being brought to the market commercially. They include enzymes, pharmaceutical proteins, antimicrobial peptides, as well as allergens of transgenic plants.

The “donor protein” is the protein that was used to raise antibodies used to identify antibody binding sequences, hence the donor protein provides the information that leads to the epitope patterns.

The “acceptor protein” is the protein, whose structure is used to fit the identified epitope patterns and/or to fit the antibody binding sequences. Hence the acceptor protein is also the parent protein.

An “autoepitope” is one that has been identified using antibodies raised against the parent protein, i.e. the acceptor and the donor proteins are identical.

A “heteroepitope” is one that has been identified with distinct donor and acceptor proteins.

The term “functionality” of protein variants refers to e.g. enzymatic activity; binding to a ligand or receptor; stimulation of a cellular response (e.g. 3H-thymidine incorporation as response to a mitogenic factor); or anti-microbial activity.

By the term “specific polyclonal antibodies” is meant polyclonal antibodies isolated according to their specificity for a certain antigen, e.g. the protein backbone.

By the term “monospecific antibodies” is meant polyclonal antibodies isolated according to their specificity for a certain epitope. Such monospecific antibodies will bind to the same epitope, but with different affinity, as they are produced by a number of antibody producing cells recognizing overlapping but not necessarily identical epitopes.

The term “randomized library” of protein variants refers to a library with at least partially randomized composition of the members, e.g. protein variants.

‘Spiked mutagenesis’ is a form of site-directed mutagenesis, in which the primers used have been synthesized using mixtures of oligonucleotides at one or more positions.

By the term “a protein variant having modified immunogenicity as compared to the parent protein” is meant a protein variant which differs from the parent protein in one or more amino acids whereby the immunogenicity of the variant is modified. The modification of immunogenicity may be confirmed by testing the ability of the protein variant to elicit an IgE/IgG response.

In the present context the term “protein” is intended to cover oligopeptides, polypeptides as well as proteins as such.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to a parent protein, comprising the steps of:

a) obtaining antibody binding peptide sequences,

b) using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein,

c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence,

d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein,

e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and

f) evaluating the immunogenicity of the protein variant using the parent protein as reference.

A) How to Find Antibody Binding Peptide Sequences and Epitope Patterns

A first step of the method is to identify peptide sequences, which bind specifically to antibodies.

Antibody binding peptide sequences can be found by testing a set of known peptide sequences for binding to antibodies raised against the donor protein. These sequences are typically selected, such that each represents a segment of the donor protein sequence (Mol. Immunol., 1992, vol. 29, pp. 1383-1389; Am. J. Resp. Cell. Mol. Biol. 2000, vol. 22, pp. 344-351). Also, randomized synthetic peptide libraries can be used to find antibody binding sequences (Slootstra et al; Molecular Diversity, 1996, vol. 2, pp. 156-164).

In a preferred method, the identification of antibody binding sequences may be achieved by screening of a display package library, preferably a phage display library. The principle behind phage display is that a heterologous DNA sequence can be inserted in the gene coding for a coat protein of the phage (WO 92/15679). The phage will make and display the hybrid protein on its surface where it can interact with specific target agents. Such target agent may be antigen-specific antibodies. It is therefore possible to select specific phages that display antibody-binding peptide sequences. The displayed peptides can be of predetermined lengths, for example 9 amino acids long, with randomized sequences, resulting in a random peptide display package library. Thus, by screening for antibody binding, one can isolate the peptide sequences that have sufficiently high affinity for the particular antibody used. The peptides of the hybrid proteins of the specific phages which bind protein-specific antibodies characterize epitopes that are recognized by the immune system.

The antibodies used for reacting with the display package are preferably IgE antibodies to ensure that the epitopes identified are IgE epitopes, i.e. epitopes inducing and binding IgE. In a preferred embodiment the antibodies are polyclonal antibodies, optionally monospecific antibodies.

For the purpose of the present invention polyclonal antibodies are preferred in order to obtain a broader knowledge about the epitopes of a protein.

It is of great importance that the amino acid sequence of the peptides presented by the display packages is long enough to represent a significant part of the epitope to be identified. In a preferred embodiment of the invention the peptides of the peptide display package library are oligopeptides having from 5 to 25 amino acids, preferably at least 8 amino acids, such as 9 amino acids. For a given length of peptide sequences (n), the theoretical number of different possible sequences can be calculated as 20n. The diversity of the package library used must be large enough to provide a suitable representation of the theoretical number of different sequences. In a phage-display library, each phage has one specific sequence of a determined length. Hence an average phage display library can express 108-1012 different random sequences, and is therefore well-suited to represent the theoretical number of different sequences.

The antibody binding peptide sequences can be further analysed by consensus alignment e.g. by the methods described by Feng and Doolittle, Meth. Enzymol., 1996, vol. 266, pp. 368-382; Feng and Doolittle, J. Mol. Evol., 1987, vol. 25, pp. 351-360; and Taylor, Meth. Enzymol., 1996, vol. 266, pp. 343-367.

This leads to identification of epitope patterns, which can assist the comparison of the linear information obtained from the antibody binding peptide sequences to the 3-o dimensional structure of the acceptor protein in order to identify epitope sequences at the surface of the acceptor protein.

B) How to Identify Epitope Sequences and Epitope Areas.

Given a number of antibody binding peptide sequences and possibly the corresponding epitope patterns, one need the 3-dimensional structure coordinates of an acceptor protein to find the epitope sequences on its surface.

These coordinates can be found in databases (NCBI: http://www.ncbi.nlm.nih.gov/), determined experimentally using conventional methods (Ducruix and Giege: Crystallization of Nucleic Acids and Proteins, IRL PRess, Oxford, 1992, ISBN 0-19-963245-6), or they can be deduced from the coordinates of a homologous protein. Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3-dimensional structures exist, definition of Structurally Conserved Regions (SCRs), assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (>3 residues) relative to the known 3-dimensional structures are known to be quite difficult to model, and structural predictions must be considered with care.

Using the coordinates and the several methods of mapping the linear information on the 3-dimensional surface are possible, as described in the examples below.

One can match each amino acid residue of the antibody binding peptide to an identical or homologous amino acid on the 3-D surface of the acceptor protein, such that amino acids that are adjacent in the primary sequence are close on the surface of the acceptor protein, with close being <5 Å, preferably <3 Å between any two atoms of the two amino acids.

Alternatively, one can define a geometric body (e.g. an ellipsoid, a sphere, or a box) of a size that matches a possible binding interface between antibody and antigen and look for a positioning of this body where it will contain most of or all the anchor amino acids.

Also, one can use the epitope patterns to facilitate identification of epitope sequences. This can be done, by first matching the anchor amino acids on the 3-D structure and subsequently looking for other elements of the antibody binding peptide sequences, which provide additional matches. If there are many residues to be matched, it is only necessary that a suitable number can be found on the 3-D structure. For example if an epitope pattern comprises 4, 5, 6, or 7 amino acids, it is only necessary that 3 matches surface elements of the acceptor protein.

In all cases, it is desirable that amino acids of the epitope sequence are surface exposed (as described below in Examples).

It is known, that amino acids that surround binding sequences can affect binding of a ligand without participating actively in the binding process. Based on this knowledge, areas covered by amino acids with potential steric effects on the epitope-antibody interaction, were defined around the identified epitope sequences. These areas are called ‘epitope areas’. Practically, all amino acids situated within 5 Å from the amino acids defining the epitope sequence were included. Preferably, the epitope area equals the epitope sequence. The accessibility criterium was not used as hidden amino acids of an epitope area also can have an effect on the adjacent amino acids of the epitope sequence.

C) How to Use the Epitope Information.

There are at least four ways to utilize the information about epitope sequences, which has been derived by the methods of this invention:

1) reduce the allergenicity of a commercial protein using protein engineering.

2) reduce the potential of commercial proteins to cross-react with environmental allergens and hence cause allergic reactions in people sensitized to the environmental allergens (or vice versa).

3) improve the immunotherapeutic effect of allergen vaccines.

4) assist characterization of clinical allergies in order to select the appropriate allergen vaccine.

Protein Engineering to Reduce the Allergenicity, Cross-Reactivity and/or Immunotherapeutic Effect of Proteins.

The methods described thus far have led to identification of epitope areas on an acceptor protein, each containing epitope sequences. These subsets of amino acids, are preferred for introducing mutations that are meant to modify the immunogenecity of the acceptor protein. An even more preferred subset of amino acids to target by mutagenesis are ‘hot spot amino acids’, which appear in several different epitope sequences, or which corresponds to anchor amino acids of the epitope patterns.

Thus, genetic engineering mutations should be designed in the epitope areas, preferably in epitope sequences, and more preferably in the ‘hot spot amino acids’.

Substitution, Deletion, Insertion

When the epitope area(s) have been identified, a protein variant exhibiting a modified immunogenicity may be produced by changing the identified epitope area of the parent protein by genetic engineering mutation of a DNA sequence encoding the parent protein.

The epitope identified may be changed by substituting at least one amino acid of the epitope area. In a preferred embodiment at least one anchor amino acid or hot spot amino acid is changed. The change will often be substituting to an amino acid of different size, hydrophilicity, and/or polarity, such as a small amino acid versus a large amino acid, a hydrophilic amino acid versus a hydrophobic amino acid, a polar amino acid versus a non-polar amino acid and a basic versus an acidic amino acid.

Other changes may be the addition/insertion or deletion of at least one amino acid of the epitope sequence, preferably deleting an anchor amino acid or a hot spot amino acid. Furthermore, an epitope pattern may be changed by substituting some amino acids, and deleting/adding other.

In the claims a position to be changed by substitution, insertion, deletion will be indicated by: “Position xx to aaa, bbb, ccc, insertion, deletion”, meaning that position xx can be substituted by the amino acid aaa, bbb, ccc or that any amino acid can be inserted after position xx or that position xx can be deleted, e.g. “Position 27 to A, D, E, insertion, deletion” means that in position 27 the amino acid can be substituted by A, D or E, or that any amino acid can be inserted after position 27, or that the amino acid in position 27 can be deleted.

When one uses protein engineering to eliminate epitopes, it is indeed possible that new epitopes are created, or existing epitopes are duplicated. To reduce this risk, one can map the planned mutations at a given position on the 3-dimensional structure of the protein of interest, and control the emerging amino acid constellation against a database of known epitope patterns, to rule out those possible replacement amino acids, which are predicted to result in creation or duplication of epitopes. Thus, risk mutations can be identified and eliminated by this procedure, thereby reducing the risk of making mutations that lead to increased rather than decreased allergenicity.

Introduction of Residues for Chemical Derivatization in Epitope Areas

In yet another embodiment, one can design the mutation, such that amino acids suitable for chemical modification are substituted for existing ones in the epitope areas. The protein variant can then be conjugated to activated polymers. Which amino acids to substitute and/or insert, depends in principle on the coupling chemistry to be applied. The chemistry for preparation of covalent bioconjugates can be found in “Bioconjugate Techniques”, Hermanson, G. T. (1996), Academic Press Inc., which is hereby incorporated as reference (see below). It is preferred to make conservative substitutions in the polypeptide when the polypeptide has to be conjugated, as conservative substitutions secure that the impact of the substitution on the polypeptide structure is limited. In the case of providing additional amino groups this may be done by substitution of arginine to lysine, both residues being positively charged, but only the lysine having a free amino group suitable as an attachment groups. In the case of providing additional carboxylic acid groups the conservative substitution may for instance be an asparagine to aspartic acid or glutamine to glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups being present on the acidic residues. In the case of providing SH-groups the conservative substitution may be done by changing threonine or serine to cysteine.

Chemical Conjugation

For chemical conjugation, the protein variant needs to be incubate with an active or activated polymer and subsequently separated from the unreacted polymer. This can be done in solution followed by purification or it can conveniently be done using the immobilized protein variants, which can easily be exposed to different reaction environments and washes.

In the case were polymeric molecules are to be conjugated with the polypeptide in question and the polymeric molecules are not active they must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person. Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), “Protein immobilisation. Fundamental and applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.

In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.

Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Am. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).

Coupling polymeric molecules to hydroxy groups is generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.

Coupling polymeric molecules to free sulfhydryl groups can be achieved with special groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.

Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.

Techniques involving coupling of electrophilically activated PEGs to the amino groups of Lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.

Tosylate is more reactive than the mesylate but also less stable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the abovementioned groups.

Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to Lysines. Essentially the same reaction can be carried out in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.

Furthermore, isocyanates and isothiocyanates may be employed, yielding ureas and thioureas, respectively.

Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (U.S. Pat. No. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds are very high but may make the hydrolysis to fast.

PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.

Furthermore, a special linker can be introduced. The most well studied being cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

Coupling of PEG to an aromatic amine followed by diazotation yields a very reactive diazonium salt, which can be reacted with a peptide in situ. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.

As some peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.

PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.

The coupling technique used in the examples is the N-succinimidyl carbonate conjugation technique descried in WO 90/13590 (Enzon).

In a preferred embodiment, the activated polymer is methyl-PEG which has been activated by N-succinimidyl carbonate as described WO 90/13590. The coupling can be carried out at alkaline conditions in high yields.

For coupling of polymers to the protein variants, it is preferred to use conditions similar to those described in WO 96/17929 and WO 99/00489 (Novo Nordisk A/S) e.g. mono or bis activated PEG's of molecular weight ranging from 100 to 5000 Da. For instance, a methyl-PEG 350 could be activated with N-succinimidyl carbonate and incubated with protein variant at a molar ratio of more than 5 calculated as equivalents of activated PEG divided by moles of lysines in the protein of interest. For coupling to immobilized protein variant, the PEG:protein ratio should be optimized such that the PEG concentration is low enough for the buffer capacity to maintain alkaline pH throughout the reaction; while the PEG concentration is still high enough to ensure sufficient degree of modification of the protein. Further, it is important that the activated PEG is kept at conditions that prevent hydrolysis (i.e. dissolved in acid or solvents) and diluted directly into the alkaline reaction buffer. It is essential that primary amines are not present other than those occurring in the lysine residues of the protein. This can be secured by washing thoroughly in borate buffer. The reaction is stopped by separating the fluid phase containing unreacted PEG from the solid phase containing protein and derivatized protein. Optionally, the solid phase can then be washed with tris buffer, to block any unreacted sites on PEG chains that might still be present.

Introduction of Consensus Sequences for Post-Translational Modifications in the Epitope Areas

In another embodiment, the mutations are designed, such that recognition sites for post-translational modifications are introduced in the epitope areas, and the protein variant is expressed in a suitable host organism capable of the corresponding post-translational modification. These post-translational modifications may serve to shield the epitope and hence lower the immunogenicity of the protein variant relative to the protein backbone. Post-translational modifications include glycosylation, phosphorylation, N-terminal processing, acylation, ribosylation and sulfatation. A good example is N-glycosylation. N-glycosylation is found at sites of the sequence Asn-Xaa-Ser, Asn-Xaa-Thr, or Asn-Xaa-Cys, in which neither the Xaa residue nor the amino acid following the tri-peptide consensus sequence is a proline (T. E. Creighton, ‘Proteins—Structures and Molecular Properties, 2nd edition, W.H. Freeman and Co., New York, 1993, pp. 91-93). It is thus desirable to introduce such recognition sites in the sequence of the backbone protein. The specific nature of the glycosyl chain of the glycosylated protein variant may be linear or branched depending on the protein and the host cells. Another example is phosphorylation: The protein sequence can be modified so as to introduce serine phosphorylation sites with the recognition sequence arg-arg-(xaa)n-ser (where n=0, 1, or 2) (SEQ ID NOS: 38 and 39), which can be phosphorylated by the cAMP-dependent kinase or tyrosine phosphorylation sites with the recognition sequence -lys/arg-(xaa)3-asp/glu-(xaa)3-tyr (SEC) ID NO: 40), which can usually be phosphorylated by tyrosine-specific kinases (T. E. Creighton, “Proteins—Structures and molecular properties”, 2nd ed., Freeman, N.Y., 1993).

Randomized Approaches to Introduce Modifications in Epitope Areas.

In order to generate protein variants, more than one amino acid residue may be substituted, added or deleted, these amino acids preferably being located in different epitope areas. In that case, it may be difficult to assess a priori how well the functionality of the protein is maintained while antigenicity is reduced, especially since the possible number of mutation-combinations becomes very large, even for a small number of mutations. In that case, it will be an advantage, to establish a library of diversified mutants each having one or more changed amino acids introduced and selecting those variants, which show good retention of function and at the same time a significant reduction in antigenicity.

A diversified library can be established by a range of techniques known to the person skilled in the art (Reetz M T; Jaeger K E, in ‘Biocatalysis—from Discovery to Application’ edited by Fessner W D, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p. 389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998). These include, but are not limited to, ‘spiked mutagenesis’, in which certain positions of the protein sequence are randomized by carring out PCR mutagenesis using one or more oligonucleotide primers which are synthesized using a mixture of nucleotides for certain positions (Lanio T, Jeltsch A, Biotechniques, Vol. 25(6), 958,962,964-965 (1998)). The mixtures of oligonucleotides used within each triplet can be designed such that the corresponding amino acid of the mutated gene product is randomized within some predetermined distribution function. Algorithms have been disclosed, which facilitate this design (Jensen L J et al., Nucleic Acids Research, Vol. 26(3), 697-702 (1998)).

In an embodiment substitutions are found by a method comprising the following steps: 1) a range of substitutions, additions, and/or deletions are listed encompassing several epitope areas (preferably in the corresponding epitope sequences, anchor amino aids, and/or hot spots), 2) a library is designed which introduces a randomized subset of these changes in the amino acid sequence into the target gene, e.g. by spiked mutagenesis, 3) the library is expressed, and preferred variants are selected. In another embodiment, this method is supplemented with additional rounds of screening and/or family shuffling of hits from the first round of screening (J. E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896, 1999) and/or combination with other methods of reducing immunogenicity by genetic means (such as that disclosed in WO 92/10755).

The library may be designed, such that at least one amino acid of the epitope area is substituted. In a preferred embodiment at least one amino acid of the epitope sequence itself is changed, and in an even more preferred embodiment, one or more hot spot amino acids are changed. The library may be biased such that towards introducing an amino acid of different size, hydrophilicity, and/or polarity relative to the original one of the ‘protein backbone’. For example changing a small amino acid to a large amino acid, a hydrophilic amino acid to a hydrophobic amino acid, a polar amino acid to a non-polar amino acid or a basic to an acidic amino acid. Other changes may be the addition or deletion of at least one amino acid of the epitope area, preferably deleting an anchor amino acid. Furthermore, substituting some amino acids and deleting or adding others may change an epitope.

Diversity in the protein variant library can be generated at the DNA triplet level, such that individual codons are variegated e.g. by using primers of partially randomized sequence for a PCR reaction. Further, several techniques have been described, by which one can create a library with such diversity at several locations in the gene, which are too far apart to be covered by a single (spiked) oligonucleotide primer. These techniques include the use of in vivo recombination of the individually diversified gene segments as described in WO 97/07205 on page 3, line 8 to 29 or by using DNA shuffling techniques to create a library of full length genes that combine several gene segments each of which are diversified e.g. by spiked mutagenesis (Stemmer, Nature 370, pp. 389-391, 1994 and U.S. Pat. Nos. 5,605,793 and 5,830,721). In the latter case, one can use the gene encoding the “protein backbone” as a template double-stranded polynucleotide and combining this with one or more single or double-stranded oligonucleotides as described in claim 1 of U.S. Pat. No. 5,830,721. The single-stranded oligonucleotides could be partially randomized during synthesis. The double-stranded oligonucleotides could be PCR products incorporating diversity in a specific region. In both cases, one can dilute the diversity with corresponding segments containing the sequence of the backbone protein in order to limit the number of changes that are on average introduced. As mentioned above, methods have been established for designing the ratios of nucleotides (A; C; T; G) used at a particular codon during primer synthesis, so as to approximate a desired frequency distribution among a set of desired amino acids at that particular codon. This allows one to bias the partially randomized mutagenesis towards e.g. introduction of post-translational modification sites, chemical modification sites, or simply amino acids that are different from those that define the epitope or the epitope area. One could also approximate a sequence in a given location or epitope area to the corresponding location on a homologous, human protein.

Occasionally, one would be interested in testing a library that combines a number of known mutations in different locations in the primary sequence of the ‘protein backbone’. These could be introduced post-translational or chemical modification sites, or they could be mutations, which by themselves had proven beneficial for one reason or another (e.g. decreasing antigenicity, or improving specific activity, performance, stability, or other characteristics). In such cases, it may be desirable to create a library of diverse combinations of known sequences. For example if 12 individual mutations are known, one could combine (at least) 12 segments of the ‘protein backbone’ gene in which each segment is present in two forms: one with and one without the desired mutation. By varying the relative amounts of those segments, one could design a library (of size 212) for which the average number of mutations per gene can be predicted. This can be a useful way of combining elements that by themselves give some, but not sufficient effect, without resorting to very large libraries, as is often the case when using ‘spiked mutagenesis’. Another way to combine these ‘known mutations’ could be by using family shuffling of oligomeric DNA encoding the known changes with fragments of the full length wild type sequence.

Assays for Reduced Allergenicity

When protein variants have been constructed based on the methods described in this invention, it is desirable to confirm their antibody binding capacity, functionality, immunogenicity and/or allergenicity using a purified preparation. For that use, the protein variant of interest can be expressed in larger scale, purified by conventional techniques, and the antibody binding and functionality should be examined in detail using dose-response curves and e.g. direct or competitive ELISA (C-ELISA).

The potentially reduced allergenicity (which is likely, but not necessarily true for a variant w. low antibody binding) should be tested in in vivo or in vitro model systems: e.g. an in vitro assays for immunogenicity such as assays based on cytokine expression profiles or other proliferation or differentiation responses of epithelial and other cells incl. B-cells and T-cells. Further, animal models for testing allergenicity should be set up to test a limited number of protein variants that show desired characteristics in vitro. Useful animal models include the guinea pig intratracheal model (GPIT) (Ritz, et al. Fund. Appl. Toxicol., 21, pp. 31-37, 1993), mouse subcutaneous (mouse-SC) (WO 98/30682, Novo Nordisk), the rat intratracheal (rat-IT) (WO 96/17929, Novo Nordisk), and the mouse intranasal (MINT) (Robinson et al., Fund. Appl. Toxicol. 34, pp. 15-24, 1996) models.

The immunogenicity of the protein variant is measured in animal tests, wherein the animals are immunised with the protein variant and the immune response is measured. Specifically, it is of interest to determine the allergenicity of the protein variants by repeatedly exposing the animals to the protein variant by the intratracheal route and following the specific IgG and IgE titers. Alternatively, the mouse intranasal (MINT) test can be used to assess the allergenicity of protein variants. By the present invention the allergenicity is reduced at least 3 times as compared to the allergenicity of the parent protein, preferably 10 times reduced, more preferably 50 times.

However, the present inventors have demonstrated that the performance in ELISA correlates closely to the immunogenic responses measured in animal tests. To obtain a useful reduction of the allergenicity of a protein, the IgE binding capacity of the protein variant must be reduced to at least below 75%, preferably below 50%, more preferably below 25% of the IgE binding capacity of the parent protein as measured by the performance in IgE ELISA, given the value for the IgE binding capacity of the parent protein is set to 100%.

Thus a first assessment of the immunogenicity and/or allergenicity of a protein can be made by measuring the antibody binding capacity or antigenicity of the protein variant using appropriate antibodies. This approach has also been used in the literature (WO 99/47680).

Assays for Altered Immunotherapeutic Effect

The immunotherapeutic effect of allergen vaccines can be assessed a number of different ways. One is to measure the specific IgE binding, the reduction of which indicates a better allergen vaccine potential (WO 99/47680, ALK-ABELLO). Also, several cellular assays could be employed to show the modified immuneresponse indicative of good allergen vaccine potential as shown in several publications, all of which are hereby incorporated by reference (van Neerven et al, “T lymphocyte responses to allergens: Epitope-specificity and clinical relevance”, Immunol Today, 1996, vol. 17, pp. 526-532; Hoffmann et al., Allergy, 1999, vol. 54, pp. 446-454, WO 99/07880).

Eventually, clinical trials with allergic patients could be employed using cellular or clinical end-point measurements. (Ebner et al., Clin. Exp. All., 1997, vol. 27, pp. 107-1015; Int. Arch. Allergy Immunol., 1999, vol. 119, pp 1-5).

Determining Functionality

A wide variety of protein functionality assays are available in the literature. Especially, those suitable for automated analysis are useful for this invention. Several have been published in the literature such as protease assays (WO 99/34011, Genencor International; J. E. Ness, et al, Nature Biotechn., 17, pp. 893-896, 1999), oxidoreductase assays (Chemy et al., Nature Biotechn., 17, pp. 379-384, 1999, and assays for several other enzymes (WO 99/45143, Novo Nordisk). Those assays that employ soluble substrates can be employed for direct analysis of functionality of immobilized protein variants.

Cross-Reactivity

A related objective is to reduce cross-reactivity between ‘commercial allergens’ and ‘environmental allergens’. Cross-reactivities between food allergens of different origin are well-known (Akkerdaas et al, Allergy 50, pp 215-220, 1995). Similarly, cross-reactivities between other environmental allergens (like pollen, dust mites etc.) and commercial allergens (like enzyme proteins) have been established in the literature (J. All. Clin. Immunol., 1998, vol. 102, pp. 679-686 and by the present inventors. The molecular reason for this cross-reactivity can be explored using epitope mapping. By finding epitope patterns using antibodies raised against environmental allergen (donor protein) and mapping this information on a commercial allergen (the acceptor protein), one may find the epitopes that are common to both proteins, and hence responsible for the cross-reactivity. Obviously, one can also use the commercial allergen as donor and the environmental allergen as acceptor. By modifying the commercial allergen using protein engineering in the epitope areas identified as described above, one can reduce the cross-reactivity of the commercial allergen variant towards the environmental allergens (and vice versa). Hence, the use of the modified commercial allergens would be safer than using the unmodified commercial allergen.

Testing of this approach would be done using an antibody-binding assay with the protein variant (and its parent protein as control) and antibodies raised against the protein that cross-reacts with the parent protein. The method is otherwise identical to those described in the Methods section for characterization of allergencitiy and antigenicity.

Wash Performance etc.

The modifications of the enzymes in the epitope areas as disclosed the present application may cause other effects to the enzyme than modified immunogenicity. A modification may also change the performance of the enzyme, such as the wash performance, thermo stability, storage stability and increased catalytical activity of the enzyme.

The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, wash-ability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.

Commercial Enzyme Applications Industrial Applications

Another aspect of the invention is a composition comprising at least one protein (polypeptide) or enzyme of the invention. The composition may comprise other polypeptides, proteins or enzymes and/or ingredients normally used in personal care products, such as shampoo, soap bars, skin lotion, skin creme, hair dye, toothpaste, household articles, agro chemicals, personal care products, such as cleaning preparations e.g. for contact lenses, cosmetics, toiletries, oral and dermal pharmaceuticals, compositions used for treating textiles, compositions used for manufacturing food, e.g. baking, and feed etc.

Examples of said proteins(polypeptides)/enzymes include enzymes exhibiting protease, lipolytic enzyme, oxidoreductase, carbohydrase, transferase, such as transglutaminase, phytase and/or anti-microbial polypeptide activity. These enzymes may be present as conjugates with reduced activity.

The protein of the invention may furthermore typically be used in detergent composition. It may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri NS) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in patent GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition may be in any convenient form, e.g. as powder, granules, paste or liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic. The detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).

The detergent composition may additionally comprise one or more other enzymes, such as e.g. proteases, amylases, lipolytic enzymes, cutinases, cellulases, peroxidases, oxidases, and further anti-microbial polypeptides.

The detergent may contain 1-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst). The detergent may also be unbuilt, i.e. essentially free of detergent builder.

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfon-ate (NOBS). Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The detergent composition of the invention comprising the polypeptide of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as, e.g., an aromatic borate ester, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, bactericides, optical brighteners, or perfume.

The pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11.

Dishwashing Composition

Further, a modified enzyme according to the invention may also be used in dishwashing detergents.

Dishwashing detergent compositions comprise a surfactant which may be anionic, non-ionic, cationic, amphoteric or a mixture of these types. The detergent will contain 0-90% of non-ionic surfactant such as low- to non-foaming ethoxylated propoxylated straight-chain alcohols.

The detergent composition may contain detergent builder salts of inorganic and/or organic types. The detergent builders may be subdivided into phosphorus-containing and non-phosphorus-containing types. The detergent composition usually contains 1-90% of detergent builders.

Examples of phosphorus-containing inorganic alkaline detergent builders, when present, include the water-soluble salts especially alkali metal pyrophosphates, orthophosphates, and polyphosphates. An example of phosphorus-containing organic alkaline detergent builder, when present, includes the water-soluble salts of phosphonates. Examples of non-phosphorus-containing inorganic builders, when present, include water-soluble alkali metal carbonates, borates and silicates as well as the various types of water-insoluble crystalline or amorphous alumino silicates of which zeolites are the best-known representatives.

Examples of suitable organic builders include the alkali metal, ammonium and substituted ammonium, citrates, succinates, malonates, fatty acid sulphonates, carboxymetoxy succinates, ammonium polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates, polyacetyl carboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weight polymers and co-polymers known to have builder properties, for example appropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acid copolymers and their salts.

The dishwashing detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type. Examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite and hypobromite as well as chlorinated trisodium phosphate. Examples of organic chlorine/bromine-type bleaches are heterocyclic N-bromo and N-chloro imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations such as potassium and sodium. Hydantoin compounds are also suitable.

The oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound. Typical examples of suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates and perphosphates. Preferred activator materials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilized using conventional stabilizing agents for the enzyme(s), e.g. a polyol such as e.g. propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition of the invention may also contain other conventional detergent ingredients, e.g. deflocculant material, filler material, foam depressors, anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bactericides, fluorescers, thickeners and perfumes.

Finally, the enzyme of the invention may be used in conventional dishwashing-detergents, e.g. in any of the detergents described in any of the following patent publications: EP 518719, EP 518720, EP 518721, EP 516553, EP 516554, EP 516555, GB 2200132, DE 3741617, DE 3727911, DE 4212166, DE 4137470, DE 3833047, WO 93/17089, DE 4205071, WO 52/09680, WO 93/18129, WO 93/04153, WO 92/06157, WO 92/08777, EP 429124, WO 93/21299, U.S. Pat. No. 5,141,664, EP 561452, EP 561446, GB 2234980, WO 93/03129, EP 481547, EP 530870, EP 533239, EP 554943, EP 346137, U.S. Pat. No. 5,112,518, EP 318204, EP 318279, EP 271155, EP 271156, EP 346136, GB 2228945, CA 2006687, WO 93/25651, EP 530635, EP 414197, and U.S. Pat. No. 5,240,632.

Personal Care Applications

A particularly useful application area for low allergenic proteins or of proteins with low cross-reactivity to environmental allergens would be in personal care products where the end-user is in close contact with the protein, and where certain problems with allergenicity has been encountered in experimental set-ups (Kelling et al., J. All. Clin. 1 mm., 1998, Vol. 101, pp. 179-187 and Johnston et al., Hum. Exp. Toxicol., 1999, Vol. 18, p. 527).

First of all the conjugate or compositions of the invention can advantageously be used for personal care products, such as hair care and hair treatment products. This include products such as shampoo, balsam, hair conditioners, hair waving compositions, hair dyeing compositions, hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.

Further contemplated are oral care products such as dentifrice, oral washes, chewing gum.

Also contemplated are skin care products and cosmetics, such as skin cream, skin milk, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, nourishing essence, skin lotion, milky lotion, calamine lotion, hand cream, powder soap, transparent soap, sun oil, sun screen, shaving foam, shaving cream, baby oil lipstick, lip cream, creamy foundation, face powder, powder eye-shadow, powder, foundation, make-up base, essence powder, whitening powder.

Also for contact lenses hygiene products the conjugate of the invention can be used advantageously. Such products include cleaning and disinfection products for contact lenses.

Proteases

Proteases are well-known active ingredients for cleaning of contact lenses. They hydrolyse the proteinaceous soil on the lens and thereby makes it soluble. Removal of the protein soil is essential for the wearing comfort.

Proteases are also effective ingredients in skin cleaning products, where they remove the upper layer of dead keratinaseous skin cells and thereby make the skin look brighter and fresher.

Proteases are also used in oral care products, especially for cleaning of dentures, but also in dentifrices.

Further, proteases are used in toiletries, bath and shower products, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.

Lipolytic Enzymes

Lipolytic enzymes can be applied for cosmetic use as active ingredients in skin cleaning products and anti-acne products for removal of excessive skin lipids, and in bath and shower products such as creams and lotions as active ingredients for skin care.

Lipolytic enzymes can also be used in hair cleaning products (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair.

Lipolytic enzymes are also effective ingredients in products for cleaning of contact lenses, where they remove lipid deposits from the lens surface.

Oxidoreductases

The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e.g. glucose) that ensures production of H2O2, which then will initiate the oxidation of for instance SON− or I− into antimicrobial reagents (SCNO− or I2) by a peroxidase (usually lactoperoxidase). This enzymatic complex is known in nature from e.g. milk and saliva.

It is being utilised commercially as anti-microbial system in oral care products (mouth rinse, dentifrice, chewing gum) where it also can be combined with an amyloglucosidase to produce the glucose. These systems are also known in cosmetic products for preservation.

Anti-microbial systems comprising the combination of an oxidase and a peroxidase are know in the cleaning of contact lenses.

Another application of oxidoreductases is oxidative hair dyeing using oxidases, peroxidases and laccases.

Free radicals formed on the surface of the skin (and hair) known to be associated with the ageing process of the skin (spoilage of the hair). The free radicals activate chain reactions that lead to destruction of fatty membranes, collagen, and cells. The application of free radical scavengers such as Superoxide dismutase into cosmetics is well known (R. L. Goldemberg, DCI, Nov. 93, p. 48-52).

Protein disulfide isomerase (PDI) is also an oxidoreductase. It can be utilised for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds).

Carbohydrases

Plaque formed on the surface of teeth is composed mainly of polysaccharides. They stick to the surface of the teeth and the microorganisms. The polysaccharides are mainly α-1,6 bound glucose (dextran) and α-1,3 bound glucose (mutan). The application of different types of glucanases such as mutanase and dextranase helps hydrolysing the sticky matrix of plaque, making it easier to remove by mechanical action.

Also other kinds of biofilm for instance the biofilm formed in lens cases can be removed by the action of glucanases.

Food and Feed

Further conjugated enzymes or polypeptides with reduced immunogenicity according to the invention may advantageously be used in the manufacturing of food and feed.

Proteases

The gluten in wheat flour is the essential ingredient responsible for the ability of flour to be used in baked foodstuffs. Proteolytic enzymes are sometimes needed to modify the gluten phase of the dough, e.g. a hard wheat flour can be softened with a protease.

Neutrase® is a commercially available neutral metallo protease that can be used to ensure a uniform dough quality and bread texture, and to improve flavour. The gluten proteins are degraded either moderately or more extensively to peptides, whereby close control is necessary in order to avoid excessive softening of the dough.

Proteases are also used for modifying milk protein.

To coagulate casein in milk when producing cheese proteases such as rennet or chymosin may be used.

In the brewery industry proteases are used for brewing with unmalted cereals and for controlling the nitrogen content.

In animal feed products proteases are used so to speak to expand the animals digestion system.

Lipolytic Enzymes

Addition of lipolytic enzyme results in improved dough properties and an improved breadmaking quality in terms of larger volume, improved crumb structure and whiter crumb colour. The observed effect can be explained by a mechanism where the lipolytic enzyme changes the interaction between gluten and some lipids fragment during dough mixing. This results in an improved gluten network.

The flavour development of blue roan cheese (e.g. Danablue), certain Italian type cheese, and other dairy products containing butter-fat, are dependent on the degradation of milk fat into free fatty acids. Lipolytic enzymes may be used for developing flavour in such products.

In the oil- and fat producing industry lipases are used e.g. to minimize the amount of undesirable side-products, to modify fats by interesterification, and to synthesis of esters.

Oxidoreductases

Further oxidoreductases with reduced immunogenicity according to the invention may advantageously be used in the manufacturing of food and feed.

Several oxidoreductases are used for baking, glucose oxidase, lipoxygenase, peroxidase, catalase and combinations hereof. Traditionally, bakers strengthen gluten by adding ascorbic acid and potassium bromate. Some oxidoreductases can be used to replace bromate in dough systems by oxidation of free sulfydryl units in gluten proteins. Hereby disulphide linkages are formed resulting in stronger, more elastic doughs with greater resistance.

Gluzyme™ (Novozymes A/S) is a glucose oxidase preparation with catalase activity that can be used to replace bromate. The dough strengthen is measured as greater resistance to mechanical shock, better oven spring and larger loaf volume.

Carbohydrases

Flour has varying content of amylases leading to differences in the baking quality. Addition of amylases can be necessary in order to standardize the flour. Amylases and pentosanases generally provide sugar for the yeast fermentation, improve the bread volume, retard retrogradation, and decrease the staling rate and stickiness that results from pentosan gums. Examples of carbohydrases are given below.

Certain maltogenic amylases can be used for prolonging the shelf life of bread for two or more days without causing gumminess in the product. Selectively modifies the gelatinized starch by cleaving from the non-reducing end of the starch molecules, low molecular wight sugars and dextrins. The starch is modified in such a way that retrogradation is less likely to occur. The produced low-molecular-weight sugars improve the baked goods water retention capacity without creating the intermediate-length dextrins that result in gumminess in the finished product. The enzyme is inactivated during bread baking, so it can be considered a processing aid that does not have to be declared on the label. Overdosing of Novamyl can almost be excluded.

The bread volume can be improved by fungal α-amylases which further provide good and uniform structure of the bread crumb. Said α-amylases are endoenzymes that produce maltose, dextrins and glucose. Cereal and some bacterial α-amylases are inactivated at temperatures above the gelatinization temperature of starch, therefore when added to wheat dough it results in a low bread volume and a sticky bread interior. Fungamyl has the advantage of being thermolabile and is inactivated just below the gelatinization temperature.

Enzyme preparations containing a number of pentosanase and hemi-cellulase activities can improve the handling and stability of the dough, and improves the freshness, the crumb structure and the volume of the bread.

By hydrolysing the pentosans fraction in flour, it will lose a great deal of its water-binding capacity, and the water will then be available for starch and gluten. The gluten becomes more pliable and extensible, and the starch gelatinizes more easily. Pentosanases can be used in combination with or as an alternative to emulsifiers.

Further carbohydrases are user for producing syrups from starch, which are widely used in soft drinks, sweets, meat products, dairy products, bread products, ice cream, baby food, jam etc.

The conversion of starch is normally carried out three steps. First the starch is liquefied, by the use of alpha-amylases. Maltodextrins, primary consisting of oligosaccharides and dextrins, are obtained.

The mixture is then treated with an amyloglucosidase for hydrolysing the oligosaccharides and dextrins into glucose. This way a sweeter product is obtained. If high maltose syrups are desired beta-amylases alone or in combination with a pullulanase (de-branching enzyme) may be used.

The glucose mixture can be made even sweeter by isomerization to fructose. For this an immobilized glucose isomerase can be used.

In the sugar industry, it is common practice to speed up the break down of present starch in cane juices. Thereby the starch content in the raw sugar is reduced and filtration at the refinery facilitated.

Furthermore dextranases are used to break down dextran in raw sugar juices and syrups.

In the alcohol industry alpha-amylases is advantageously being used for thinning of starch in distilling mashes.

In the brewing industry alpha-amylases is used for adjunct liquefaction.

In the dairy industry beta-galactosidases (lactase) is used when producing low lactose milk for persons suffering from lactose malabsorption.

When flavoured milk drinks are produced from lactase-treated milk, the addition of sugar can be reduced without reducing the sweetness of the product.

In the production of condensed milk, lactose crystallization can be avoided by lactase treatment, and the risk of thickening caused by casein coagulation in lactose crystals is thus reduced.

When producing ice cream made from lactase-treated milk (or whey) no lactose crystals will be formed and the defect, sandiness, will not occur.

Further, xylanases are known to be used within a number of food/feed industrial applications as described in WO 94/21785 (Novo Nordisk A/S).

Alpha-amylases are used in the animal feed industry to be added to cereal-containing feed to improve the digestibility of starch.

Anti-Microbial Polypeptides

Certain bacteriolytic enzymes may be used e.g. to wash carcasses in the meat packing industry (see U.S. Pat. No. 5,354,681 from Novo Industri A/S)

Transferases

Transglutaminases with reduced immunogenicity according to the invention may advantageously be used in the manufacturing of food and feed.

Transglutaminases has the ability to crosslinking protein.

This property can be used for gelling of aqueous phases containing proteins. This may be used for when producing of spreads (DK patent application no. 1071/84 from Novo Nordisk A/S).

Transglutaminases are being used for improvement of baking quality of flour e.g. by modifying wheat flour to be used in the preparation of cakes with improved properties, such as improved taste, dent, mouth-feel and a higher volume (see JP 1-110147).

Further producing paste type food material e.g. used as fat substitution in foods as ice cream, toppings, frozen desserts, mayonnaises and low fat spreads (see WO 93/22930 from Novo Nordisk A/S).

Furthermore for preparation of gels for yoghurt, mousses, cheese, puddings, orange juice, from milk and milk-like products, and binding of chopped meat product, improvement of taste and texture of food proteins (see WO 94/21120 and WO 94/21129 from Novo Nordisk A/S).

Phytases

Phytases of the invention may advantageously be used in the manufacturing of food, such as breakfast cereal, cake, sweets, drinks, bread or soup etc., and animal feed.

Phytases may be used either for exploiting the phosphorus bound in the phytate/phytic acid present in vegetable protein sources or for exploiting the nutritionally important minerals bound in phytic acid complexes.

Microbial phytase may be added to feedstuff of monogastric animals in order to avoid supplementing the feed with inorganic phosphorus (see U.S. Pat. No. 3,297,548).

Further phytases may be used in soy processing. Soyabean meal may contain high levels of the anti-nutritional factor phytate which renders this protein source unsuitable for application in baby food and feed for fish, calves and other non-ruminants, since the phytate chelates essential minerals present therein (see EP 0 420 358).

Also for baking purposes phytases may be used. Bread with better quality can be prepared by baking divided pieces of a dough containing wheat flour etc. and phytase (see JP-0-3076529-A).

A high phytase activity as in koji mold are known to be used for producing refined sake (see JP-0-6070749-A).

Textile Applications Proteases

Proteases are used for degumming and sand washing of silk.

Lipolytic Enzymes

Lipolytic enzymes are used for removing fatty matter containing hydrophobic esters (e.g. triglycerides) during the finishing of textiles (see e.g. WO 93/13256 from Novo Nordisk A/S).

Oxidoreductases

In bleach clean up of textiles catalases may serve to remove excess hydrogen peroxide.

Carbohydrases

Cellulolytic enzymes are widely used in the finishing of denim garments in order to provide a localized variation in the colour density of the fabric (Enzyme facilitated “stone wash”).

Also cellulolytic enzymes find use in the bio-polishing process. Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance without loss of fabric wettability. Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278.

During the weaving of textiles, the threads are exposed to considerable mechanical strain. In order to prevent breaking, the threads are usually reinforced by the coating (sizing) with a gelatinous substance (size). The most common sizing agent is starch in native or modified form. A uniform and durable finish can thus be obtained only after removal of the size from the fabric, the so-called desizing. Desizing of fabrics sized with a size containing starch or modified starch is preferably facilitated by use of amylolytic enzymes.

Oral and Dermal Pharmaceuticals Proteases

Different combinations of highly purified proteases (e.g. Trypsin and Chymotrypsin) are used in pharmaceuticals to be taken orally, and dermal pharmaceuticals for combating e.g. inflammations, edemata and injuries.

Leather Production Transferase

Transglutaminase is known to be used to casein-finishing leather by acting as a hardening agent (see WO 94/13839 from Novo Nordisk).

Hard Surface Cleaning

Cleaning of hard surfaces e.g. in the food industry is often difficult, as equipment used for producing dairies, meat, sea food products, beverages etc. often have a complicated shape. The use of surfactant compositions in the form gels and foams comprising enzymes have shown to facilitate and improve hard surface cleaning. Enzymes, which advantageously may be added in such surfactant compositions, are in particular proteases, lipolytic enzymes, amylases and cellulases.

Such hard surface cleaning compositions comprising enzymes may also advantageously be used in the transport sector, for instance for washing cars and for general vessel wash.

Furthermore this invention relates to the method by which the protein variants are being synthesised and expressed in host cells. This is achieved by culturing host cells capable of expressing a polypeptide in a suitable culture medium to obtain expression and secretion of the polypeptide into the medium, followed by isolation of the polypeptide from the culture medium. The host cell may be any cell suitable for the large-scale production of proteins, capable of expressing a protein and being transformed by an expression vector.

The host cell comprises a DNA construct as defined above, optionally the cells may be transformed with an expression vector comprising a DNA construct as defined above. The host cell is selected from any suitable cell, such as a bacterial cell, a fungal cell, an animal cell, such as an insect cell or a mammalian cell, or a plant cell.

Immunotherapy

A number of vaccination approaches have been described to for infective diseases as well as for non-infective diseases (such as cancers). In a number of cases, the antigen provided is an isolated protein or protein-adjuvant mixture and more and more often, the protein is recombinant (e.g. the hepatitits B vaccine from Merck & Co). In these cases, it could be desirable to modify the immunogenicity of the antigen vaccine, such that it offers a stronger or more specific protection. This can be achieved by protein engineering of the amino acid sequence of the antigen, and would be greatly facilitated by the use of the methods of this invention for identification of epitopes on the antigen vaccine to be the favored sites for modification.

There are several examples of vaccine molecules that have been engineered to achieve a specific immune protection against virus, parasites or cancer (Ryu and Nam, Biotechnol. Prog., 2000, vol. 16 pp. 2-16; and references cited therein). “The goal is often to vaccinate with a minimal strucutre consisting of a well-defined antigen, to stimulate an effective specific immune response, while avoiding potentially hazardous risks” (Ryu and Nam, Biotechnol. Prog., 2000, vol. 16 pp. 2-16). Thus, the methods of this invention can be used to identify such minimal structures that define an antigen (or epitope thereof) whether in the form of the parent protein scaffold with a number of mutations introduced in it, or whether it is in the form of the antibody binding peptides themselves.

Allergen Vaccines

Today, a patient suffering allergic disease may be subjected to allergy vaccine therapy using allergens selected on the basis of testing the specificity of the patient's serum IgE against a bank of allergen extracts (or similar specificity tests of the patient's sensibilization such as skin prick test.

One could improve the quality of characterization by using antibody binding peptides corresponding to various epitope sequences on the protein allergens of interest. This would require a kit comprising reagents for such specificity characterization, e.g. the antibody binding peptides of desired specificity. It would be preferred to use antibody binding sequences in the kit, which correspond to defined epitope sequences known to be specific for the allergen under investigation (i.e. not identified on other allergens and/or not cross-reacting with sera raised against other allergens). This kit would be useful to specifying which allergy the patient is suffering from. This kit will lead to a more specific answer than those kits used today, and hence to a better selection of allergen vaccine therapy for the individual patient.

Further, the knowledge about cross-reacting epitopes may improve vaccine development.

In an extension of this approach, one could also characterize the patient's serum by identifying the corresponding antibody binding peptides among a random display library using the aforementioned methods. This again may lead to a better selection of allergen vaccine therapy.

Further, one could use the individual antibody binding sequences as allergen vaccines leading to more specific allergen vaccine. These antibody binding sequences could be administered in an isolated form or fused to a membrane protein of the phage display system, or to another protein, which may have beneficial effect for the immunoprotective effect of the antibody binding peptide (Dalum et al., Nature Biotechnology, 1999, Vol. 17, pp. 666-669).

D) Variations Possible Parent Protein

The “parent protein” can in principle be any protein molecule of biological origin, non-limiting examples of which are peptides, polypeptides, proteins, enzymes, post-translationally modified polypeptides such as lipopeptides or glycosylated peptides, anti-microbial peptides or molecules, and proteins having pharmaceutical properties etc.

Accordingly the invention relates to a method, wherein the “parent protein” is chosen from the group consisting of polypeptides, small peptides, lipopeptides, antimicrobials, and pharmaceutical polypeptides.

The term “pharmaceutical polypeptides” is defined as polypeptides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body of humans and/or animals.

Pharmaceutical polypeptides are potentially immunogenic as they are introduced into the circulatory system.

Examples of “pharmaceutical polypeptides” contemplated according to the invention include insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO) and prolactin.

However, the proteins are preferably to be used in industry, housekeeping and/or medicine, such as proteins used in personal care products (for example shampoo; soap; skin, hand and face lotions; skin, hand and face cremes; hair dyes; toothpaste), food (for example in the baking industry), detergents and pharmaceuticals.

Antimicrobial Peptides.

The antimicrobial peptide (AMP) may be, e.g., a membrane-active antimicrobial peptide, or an antimicrobial peptide affecting/interacting with intracellular targets, e.g. binding to cell DNA. The AMP is generally a relatively short peptide, consisting of less than 100 amino acid residues, typically 20-80 residues. The antimicrobial peptide has bactericidal and/or fungicidal effect, and it may also have antiviral or antitumour effects. It generally has low cytotoxicity against normal mammalian cells.

The antimicrobial peptide is generally highly cationic and hydrophobic. It typically contains several arginine and lysine residues, and it may not contain a single glutamate or asparatate. It usually contains a large proportion of hydrophobic residues. The peptide generally has an amphiphilic structure, with one surface being highly positive and the other hydrophobic.

The bioactive peptide and the encoding nucleotide sequence may be derived from plants, invertebrates, insects, amphibians and mammals, or from microorganisms such as bacteria and fungi.

The antimicrobial peptide may act on cell membranes of target microorganisms, e.g. through nonspecific binding to the membrane, usually in a membrane-parallel orientation, interacting only with one face of the bilayer.

The antimicrobial peptide typically has a structure belonging to one of five major classes: a helical, cystine-rich (defensin-like), b-sheet, peptides with an unusual composition of regular amino acids, and peptides containing uncommon modified amino acids.

Examples of alpha-helical peptides are Magainin 1 and 2; Cecropin A, B and P1; CAP18; Andropin; Clavanin A or AK; Styelin D and C; and Buforin II. Examples of cystine-rich peptides are a-Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3; b-Defensin-12, Drosomycin, g1-purothionin, and Insect defensin A. Examples of b-sheet peptides are Lactoferricin B, Tachyplesin I, and Protegrin PG1-5. Examples of peptides with an unusual composition are Indolicidin; PR-39; Bactenicin Bac5 and Bac7; and Histatin 5. Examples of peptides with unusual amino acids are Nisin, Gramicidin A, and Alamethicin.

Another example is the antifungal peptide (AFP) from Aspergillus giganteus. As explained in detail in WO 94/01459, which is hereby incorporated by reference, the antifungal polypeptide having the amino acid sequence shown in FIG. 1 has been found in several strains of the fungal species A. giganteus, an example of which is the A. giganteus strain deposited with the Centraallbureau voor Schimmelcultures (CBS) under the deposition number CBS 526.65.

However, the antifungal polypeptide, or variants thereof, suitable for the use according to the invention are expected to be derivable from other fungal species, especially other Aspergillus species such as A. pallidus, A. clavatus, A. longivesica, A. rhizopodus and A. clavatonanicus, because of the close relationship which exists between these species and A. giganteus.

In one embodiment of the invention the protein is an enzyme, such as glycosyl hydrolases, carbohydrases, peroxidases, proteases, lipolytic enzymes, phytases, polysaccharide lyases, oxidoreductases, transglutaminases and glycoseisomerases, in particular the following.

Parent Proteases

Parent proteases (i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include proteases within this group.

Examples include proteases selected from those classified under the Enzyme Classification (E.C.) numbers:

3.4.11 (i.e. so-called aminopeptidases), including 3.4.11.5 (Prolyl aminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10 (Bacterial leucyl aminopeptidase), 3.4.11.12 (Thermophilic aminopeptidase), 3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17 (Tryptophanyl aminopeptidase), 3.4.11.18 (Methionyl aminopeptidase).

3.4.21 (i.e. so-called serine endopeptidases), including 3.4.21.1 (Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.25 (Cucumisin), 3.4.21.32 (Brachyurin), 3.4.21.48 (Cerevisin) and 3.4.21.62 (Subtilisin);

3.4.22 (i.e. so-called cysteine endopeptidases), including 3.4.22.2 (Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7 (Asclepain), 3.4.22.14 (Actimidain), 3.4.22.30 (Caricain) and 3.4.22.31 (Ananain);

3.4.23 (i.e. so-called aspartic endopeptidases), including 3.4.23.1 (Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 (Penicillopepsin) and 3.4.23.25 (Saccharopepsin); and

3.4.24 (i.e. so-called metalloendopeptidases), including 3.4.24.28 (Bacillolysin).

Serine Proteases

A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 “Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).

The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).

Subtilases

A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al., (1997).

Savinase-Like Subtilisin

One subgroup of the subtilases may be classified as savinase-like subtilisins, having at least 81% homology to Savinase, preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 96% homology, most preferably at least 98% homology to Savinase.

Parent Subtilase

The term “parent subtilase” describes a subtilase defined according to Siezen et al. (1991 and 1997). For further details see description of “SUBTILASES” immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications have been made while retaining the characteristic of a subtilase. Furthermore, a parent subtilase may also be a subtilase which has been prepared by the DNA shuffling technique, such as described by J. E. Ness et al., Nature Biotechnology, 17, 893-896 (1999).

Alternatively the term “parent subtilase” may be termed “wild type subtilase”.

Modification(s) of a Subtilase Variant

The term “modification(s)” used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.

Subtilase Variant

In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.

Examples of relevant subtilisins comprise subtilisin BPN′, subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, PD498 (WO 93/24623), thermitase, aqualysin, Bacillus PB92 protease, proteinase K, Protease TW7, and Protease TW3.

Preferred commercially available protease enzymes include Alcalase™, Savinase™ Primase™, Duralase™, Neutrase®, Dyrazym®, Esperase™, Pyrase®, Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro, Clear-Lens Pro, and Relase® (Novozymes A/S), Maxatase™ Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™ (Genencor International Inc.).

It is to be understood that also protease variants are contemplated as the parent protease. Examples of such protease variants are disclosed in EP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomas et al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol. Biol., 193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500, WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo Nordisk A/S), WO 91/00345 (Novo Nordisk A/S), EP 525 610 (Solvay) and WO 94/02618 (Gist-Brocades N.V.).

The activity of proteases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 5.

Parent Lipolytic Enzymes

Lipolytic enzymes are classified in EC 3.1.1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature (available at http://www.chem.qmw.ac.uk/iubmb/enzyme). The lipolytic enzyme may have a substrate specificity with an activity such as EC 3.1.1.3 triacylglycerol lipase, EC 3.1.1.4 phospholipase A2, EC 3.1.1.5 lysophospholipase, EC 3.1.1.26 galactolipase, EC 3.1.1.32 phospholipase A1, EC 3.1.1.73 feruloyl esterase or EC 3.1.1.74 cutinase.

The parent lipolytic enzyme may be prokaryotic, particularly a bacterial enzyme, e.g. from Pseudomonas. Examples are Pseudomonas lipases, e.g. from P. cepacia (U.S. Pat. No. 5,290,694, pdb file 1OIL), P. glumae (N Frenken et al. (1992), Appl. Envir. Microbiol. 58 3787-3791, pdb files 1TAH and 1QGE), P. pseudoalcaligenes (EP 334 462) and Pseudomonas sp. strain SD 705 (FERM BP-4772) (WO 95/06720, EP 721 981, WO 96/27002, EP 812 910). The P. glumae lipase sequence is identical to the amino acid sequence of Chromobacterium viscosum (DE 3908131 A1). Other examples are bacterial cutinases, e.g. from Pseudomonas such as P. mendocina (U.S. Pat. No. 5,389,536) or P. putida (WO 88/09367).

Alternatively, the parent lipolytic enzyme may be eukaryotic, e.g. a fungal lipolytic enzyme such as lipolytic enzymes of the Humicola family and the Zygomycetes family and fungal cutinases.

Examples of fungal cutinases are the cutinases of Fusarium solani pisi (S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)) and Humicola insolens (U.S. Pat. No. 5,827,719).

The parent lipolytic enzyme may be fungal and may have an amino acid sequence that can be aligned with SEQ ID NO: 1 which is the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438 for the lipase from Thermomyces lanuginosus (synonym Humicola lanuginosa), described in EP 258 068 and EP 305 216 (trade name LIPOLASE). The parent lipolytic enzyme may particularly have an amino acid sequence with at least 50% homology with SEQ ID NO: 1. In addition to the lipase from T. lanuginosus, other examples are a lipase from Penicillium camembertii (P25234), a lipase from Fusasrium, lipase/phospholipase from Fusarium oxysporum (EP 130064, WO 98/26057), lipase from F. heterosporum (R87979), lysophospholipase from Aspergillus foetidus (W33009), phospholipase A1 from A. oryzae (JP-A 10-155493), lipase from A. oryzae (D85895), lipase/ferulic acid esterase from A. niger (Y09330), lipase/ferulic acid esterase from A. tubingensis (Y09331), lipase from A. tubingensis (WO 98/45453), lysophospholipase from A. niger (WO 98/31790), lipase from F. solanii having an isoelectric point of 6.9 and an apparent molecular weight of 30 kDa (WO 96/18729).

Other examples are the Zygomycetes family of lipases comprising lipases having at least 50% homology with the lipase of Rhizomucor miehei (P19515. This family also includes the lipases from Absidia reflexa, A. sporophora, A. corymbifera, A. blakesleeana, A. griseola (all described in WO 96/13578 and WO 97/27276) and Rhizopus oryzae (P21811). Numbers in parentheses indicate publication or accession to the EMBL, GenBank, GeneSeqp or Swiss-Prot databases.

Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are incorporated herein by reference:

Humicola, e.g. H. brevispora, H. brevis var. thermoidea.

Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps. fluorescens (WO 89/04361), or Ps. plantarii or Ps. gladioli (U.S. Pat. No. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps. pseudoalcaligenes (EP 218 272) or.

Candida, e.g. C. cylindracea (also called C. rugosa) or C. antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO 89/02916).

Geotricum, e.g. G. candidum (Schimada et al., (1989), J. Biochem., 106, 383-388).

Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R. niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem 56, 716-719) or R. oryzae.

Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992) or B. pumilus (WO 91/16422).

Specific examples of readily available commercial lipases include Lipolase® (WO 98/35026) Lipolase™ Ultra, Lipozyme®, Palatase®, Novozym® 435, Lecitase® (all available from Novozymes A/S).

Examples of other lipases are Lumafast™, Ps. mendocian lipase from Genencor Int. Inc.; Lipomax™, Ps. pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) from Unilever; Bacillus sp. lipase from Solvay enzymes. Other lipases are available from other companies.

It is to be understood that also lipase variants are contemplated as the parent enzyme. Examples of such are described in e.g. WO 93/01285 and WO 95/22615.

The activity of the lipase can be determined as described in “Methods of Enzymatic Analysis”, Third Edition, 1984, Verlag Chemie, Weinhein, vol. 4, or as described in AF 95/5 GB (available on request from Novozymes A/S).

Parent Oxidoreductases

Parent oxidoreductases (i.e. enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include oxidoreductases within this group.

Examples include oxidoreductases selected from those classified under the Enzyme Classification (E.C.) numbers:

Glycerol-3-phosphate dehydrogenase (NAD) (1.1.1.8), Glycerol-3-phosphate dehydrogenase [NAD(P)] (1.1.1.94), Glycerol-3-phosphate 1-dehydrogenase [NADP] (1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5), Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alanine dehydrogenase (1.4.1.1), Glutamate dehydrogenase (1.4.1.2), Glutamate dehydrogenase [NAD(P)] (1.4.1.3), Glutamate dehydrogenase (NADP) (1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase (1.4.1.7), Valine dehydrogenase (NADP) (1.4.1.8), Leucine dehydrogenase (1.4.1.9), Glycine dehydrogenase (1.4.1.10), L-Amino-acid oxidase (1.4.3.2.), D-Amino-acid oxidase(1.4.3.3), L-Glutamate oxidase (1.4.3.11), Protein-lysine 6-oxidase (1.4.3.13), L-lysine oxidase (1.4.3.14), L-Aspartate oxidase (1.4.3.16), D-amino-acid dehydrogenase (1.4.99.1), Protein disulfide reductase (1.6.4.4), Thioredoxin reductase (1.6.4.5), Protein disulfide reductase (glutathione) (1.8.4.2), Laccase (1.10.3.2), Catalase (1.11.1.6), Peroxidase (1.11.1.7), Lipoxygenase (1.13.11.12), Superoxide dismutase (1.15.1.1).

Said glucose oxidases may be derived from Aspergillus niger.

Said laccases may be derived from Polyporus pinsitus, Myceliophthora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera. Because of the homology found between the above mentioned laccases (see WO 98/38287), they are considered to belong to the same class of laccases, namely the class of “Coprinus-like laccases”. Accordingly, in the present context, the term “Coprinus-like laccase” is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 50% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 55% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 60% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 65% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 70% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 75% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 80% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 85% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, or at least 90% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3, at least 95% and less than 100% or at least 98% and less than 100% to the Coprinus cinereus laccase SEQ ID NO: 3.

Bilirubin oxidases may be derived from Myrothechecium verrucaria.

The peroxidase may be derived from e.g. Soy bean, Horseradish or Coprinus cinereus.

The protein disulfide reductase may be any of the mentioned in Danish application nos. 768/93, 265/94 and 264/94 (Novo Nordisk A/S), which are hereby incorporated as references, including Protein Disulfide reductases of bovine origin, Protein Disulfide reductases derived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived from Escherichia coli.

Specific examples of readily available commercial oxidoreductases include Gluzyme™ (enzyme available from Novozymes A/S). However, other oxidoreductases are available from others.

It is to be understood that also variants of oxidoreductases are contemplated as the parent enzyme.

The activity of oxidoreductases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 3.

Parent Carbohydrases

Parent carbohydrases may be defined as all enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five and six member ring structures (i.e. enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)). Also included in the group of carbohydrases according to the invention are enzymes capable of isomerizing carbohydrates e.g. six member ring structures, such as D-glucose to e.g. five member ring structures like D-fructose.

Examples include carbohydrases selected from those classified under the Enzyme Classification (E.C.) numbers: alpha-amylase (3.2.1.1), beta-amylase (3.2.1.2), glucan 1,4-alpha-glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1,3(4)-beta-glucanase (3.2.1.6), endo-1,4-beta-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17), beta-glucosidase (3.2.1.21), alpha-galactosidase (3.2.1.22), beta-galactosidase (3.2.1.23), amylo-1,6-glucosidase (3.2.1.33), xylan 1,4-beta-xylosidase (3.2.1.37), glucan endo-1,3-beta-D-glucosidase (3.2.1.39), alpha-dextrin endo-1,6-glucosidase (3.2.1.41), sucrose alpha-glucosidase (3.2.1.48), glucan endo-1,3-alpha-glucosidase (3.2.1.59), glucan 1,4-beta-glucosidase (3.2.1.74), glucan endo-1,6-beta-glucosidase (3.2.1.75), arabinan endo-1,5-alpha-arabinosidase (3.2.1.99), lactase (3.2.1.108), chitonanase (3.2.1.132) and xylose isomerase (5.3.1.5).

Examples of relevant carbohydrases include alpha-1,3-glucanases derived from Trichoderma harzianum; alpha-1,6-glucanases derived from a strain of Paecilomyces; beta-glucanases derived from Bacillus subtilis; beta-glucanases derived from Humicola insolens; beta-glucanases derived from Aspergillus niger, beta-glucanases derived from a strain of Trichoderma; beta-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1,4-alpha-D-glucosidases (glucoamylases) derived from Aspergillus niger; alpha-amylases derived from Bacillus subtilis; alpha-amylases derived from Bacillus amyloliquefaciens; alpha-amylases derived from Bacillus stearothermophilus; alpha-amylases derived from Aspergillus oryzae; alpha-amylases derived from non-pathogenic microorganisms; alpha-galactosidases derived from Aspergillus niger, Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulases derived from Humicola insolens; cellulases derived from Trichoderma reesei; cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger, dextranases derived from Penicillium lilacinum; endo-glucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus; beta-galactosidases derived from Kluyveromyces fragilis; xylanases derived from Trichoderma reesei.

Specific examples of readily available commercial carbohydrases include Alpha-Gal™ Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™ Plus, Novozyme® 188, Carezyme® (SEQ ID NO: 5), Celluclast®, Cellusoft®, Ceremyl®, Citrozym™, Denimax™ Dezyme™, Dextrozyme™, Finizym®, Fungamyl™, Gamanase™, Glucanex®, Lactozym®, Maltogenase™, Pentopan™, Pectinex™, Promozyme®, Pulpzyme™, Novamyl™, Termamyl®, AMG (Amyloglucosidase Novo), Maltogenase®, Sweetzyme®, Aquazym®, Natalase® (SEQ ID NO: 4), SP722, AA560 (all enzymes available from Novozymes A/S). Other carbohydrases are available from other companies.

The parent cellulase is preferably a microbial cellulase. As such, the cellulase may be selected from bacterial cellulases, e.g. Pseudomonas cellulases or Bacillus, such as the Bacillus strains described in U.S. Pat. No. 4,822,516, U.S. Pat. No. 5,045,464 or EP 468 464, or B. lautus (cf. WO 91/10732), cellulases. More preferably, the parent cellulases may be a fungal cellulase, in particular Humicola, Trichoderma, Irpex, Aspergillus, Penicillium, Myceliophthora or Fusarium cellulases. Examples of suitable parent cellulases are described in, e.g. WO 91/17244. Examples of suitable Trichoderma cellulases are those described in T. T. Teeri, Gene 51, 1987, pp. 43-52. Preferably, the parent cellulase is selected from the cellulases classified in family 45, e.g. the enzymes EG B (Pseudomonas fluorescens) and EG V (Humicola insolens), as described in Henrissat, B. et al.: Biochem. J. (1993), 293, p. 781-788.

The Termamyl-Like Alpha-Amylase

It is well known that a number of alpha-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 4 of WO 00/29560 (commercially available as Termamyl®) has been found to be about 89% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 5 of WO 00/29560 and about 79% homologous with the B. stearothermophilus alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 3 of WO 00/29560. Further homologous alpha-amylases include an alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.

Still further homologous alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis alpha-amylases are Optitherm® and Takatherm® (available from Solvay), Maxamyl® (available from Gist-brocades/Genencor), Spezym AA® and Spezyme Delta AA™ (available from Genencor), and Keistase® (available from Daiwa).

Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-like alpha-amylase” is intended to indicate an alpha-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl®, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4 (WO 00/29560). In other words, a Termamyl-like alpha-amylase is an alpha-amylase which has the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 of WO 00/29560, and the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7 of WO 00/29560) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 8 of WO 00/29560) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in SEQ ID NO: 6 of WO 00/29560) or i) which displays at least 60% homology (identity), preferred at least 70%, more preferred at least 75%, even more preferred at least 80%, especially at least 85%, especially preferred at least 90%, especially at least 95%, even especially more preferred at least 97%, especially at least 99% homology with at least one of said amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 of WO 00/29560 and/or ii) displays immunological cross-reactivity with an antibody raised against one or more of said alpha-amylases, and/or iii) is encoded by a DNA sequence which hybridizes, under the low to very high stringency conditions (said conditions described below) to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 9, 10, 11, 12, and 32, respectively, of the present application (which encodes the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, and 5 herein, respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in SEQ ID NO: 13 herein and encodes the amino acid sequence shown in SEQ ID NO: 8 herein) and from SEQ ID NO: 5 of WO 95/26397 (shown in SEQ ID NO: 14 herein), respectively.

In connection with property i), the “homology” (identity) may be determined by use of any conventional algorithm, preferably by use of the gap progamme from the GCG package version 8 (August 1994) using default values for gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1 (Genetic Computer Group (1991) Programme Manual for the GCG Package, version 8, 575 Science Drive, Madison, Wis., USA 53711).

The parent Termamyl-like alpha-amylase backbone may in an embodiment have an amino acid sequence which has a degree of identity to SEQ ID NO: 4 (WO 00/29560) of at least 65%, preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99% identity determined as described above.

A structural alignment between Termamyl® (SEQ ID NO: 4) and a Termamyl-like alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha-amylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penelties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Parent Glucoamylases

Parent glucoamylase contemplated according to the present invention include fungal glucoamylases, in particular fungal glucoamylases obtainable from an Aspergillus strain, such as an Aspergillus niger or Aspergillus awamori glucoamylases and variants or mutants thereof, homologous glucoamylases, and further glucoamylases being structurally and/or functionally similar to SEQ ID NO: 2 (WO 00/04136). Specifically contemplated are the Aspergillus niger glucoamylases G1 and G2 disclosed in Boel et al. (1984), “Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs”, EMBO J. 3 (5), p. 1097-1102. The G2 glucoamylase is disclosed in SEQ ID NO: 2 (WO 00/04136). The G1 glucoamylase is disclosed in SEQ ID NO: 13 (WO 00/04136). Another AMG backbone contemplated is Talaromyces emersonii, especially Talaromyces emersonii DSM disclosed in WO 99/28448 (Novo Nordisk).

The homology referred to above of the parent glucoamylase is determined as the degree of identity between two protein sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, p. 443-453). Using Gap with the following settings for polypeptide sequence comparison: Gap creation penalty of 3.0 and Gap extension penalty of 0.1, the mature part of a polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity preferably of at least 60%, such as 70%, at least 80%, at least 90%, more preferably at least 95%, more preferably at least 97%, and most preferably at least 99% with the mature part of the amino acid sequence shown in SEQ ID NO: 2 (WO 00/04136).

Preferably, the parent glucoamylase comprise the amino acid sequences of SEQ ID NO: 2 (WO 00/04136); or allelic variants thereof; or fragments thereof that has glucoamylase activity.

A fragment of SEQ ID NO: 2 is a polypeptide which have one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. For instance, the AMG G2 (SEQ ID NO: 2) is a fragment of the Aspergillus niger G1 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102) having glucoamylase activity. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

It is to be understood that also carbohydrase variants are contemplated as the parent enzyme.

The activity of carbohydrases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 4.

Parent Transferases

Parent transferases (i.e. enzymes classified under the Enzyme Classification number E.C. 2 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include transferases within this group.

The parent transferases may be any transferase in the subgroups of transferases: transferases transferring one-carbon groups (E.C. 2.1); transferases transferring aldehyde or residues (E.0 2.2); acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4); transferases transferring alkyl or aryl groups, other that methyl groups (E.C. 2.5); transferases transferring nitrogeneous groups (2.6).

In a preferred embodiment the parent transferase is a transglutaminase E.C 2.3.2.13 (Protein-glutamine μ-glutamyltransferase).

Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the epsilon-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transferases form intramolecular or intermolecular gamma-glutamyl-epsilon-lysyl crosslinks.

Examples of transglutaminases are described in the pending DK patent application no. 990/94 (Novo Nordisk A/S).

The parent transglutaminase may be of human, animal (e.g. bovine) or microbial origin.

Examples of such parent transglutaminases are animal derived Transglutaminase, FXIIIa; microbial transglutaminases derived from Physarum polycephalum (Klein et al., Journal of Bacteriology, Vol. 174, p. 2599-2605); transglutaminases derived from Streptomyces sp., including Streptomyces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Streptoverticillium sp., including Streptoverticillium mobaraense, Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (Motoki et al., U.S. Pat. No. 5,156,956; Andou et al., U.S. Pat. No. 5,252,469; Kaempfer et al., Journal of General Microbiology, Vol. 137, p. 1831-1892; Ochi et al., International Journal of Sytematic Bacteriology, Vol. 44, p. 285-292; Andou et al., U.S. Pat. No. 5,252,469; Williams et al., Journal of General Microbiology, Vol. 129, p. 1743-1813).

It is to be understood that also transferase variants are contemplated as the parent enzyme.

The activity of transglutaminases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10.

Parent Phytases

Parent phytases are included in the group of enzymes classified under the Enzyme Classification number E.C. 3.1.3 (Phosphoric Monoester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)).

Phytases are enzymes produced by microorganisms which catalyse the conversion of phytate to inositol and inorganic phosphorus

Phytase producing microorganisms comprise bacteria such as Bacillus subtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomyces cerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species).

Examples of parent phytases include phytases selected from those classified under the Enzyme Classification (E.C.) numbers: 3-phytase (3.1.3.8) and 6-phytase (3.1.3.26).

The activity of phytases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10, or may be measured according to the method described in EP-A1-0 420 358, Example 2A.

Lyases

Suitable lyases include Polysaccharide lyases: Pectate lyases (4.2.2.2) and pectin lyases (4.2.2.10), such as those from Bacillus licheniformis disclosed in WO 99/27083.

Isomerases Protein Disulfide Isomerase

Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S) and suitable glucose isomerases include those described in Biotechnology Letter, Vol. 20, No 6, June 1998, pp. 553-56.

Contemplated isomerases include xylose/glucose Isomerase (5.3.1.5) including Sweetzyme®.

Environmental Allergens

The environmental allergens that are of interest for epitope mapping include allergens from pollen, dust mites, mammals, venoms, fungi, food items, and other plants.

Pollen, allergens include but are not limited to those of the order Fagales, Oleales, Pinales, Poales, Asterales, and Urticales; including those from Betula, Alnus, Corylus, Carpinus, Olea, Phleum pratense and Artemisia vulgaris, such as Aln g1, Cor a1, Car b1, Cry j1, Amb a1 and a2, Art v1, Par j1, Ole e1, Ave v1, and Bet v1 (WO 99/47680).

Mite allergens include but are not limited to those from Derm. farinae and Derm. pteronys., such as Der f1 and f2, and Der p1 and p2.

From mammals, relevant environmental allergens include but are not limited to those from cat, dog, and horse as well as from dandruff from the hair of those animals, such as Fel d1; Can f1; Equ c1; Equ c2; Equ c3.

Venum allergens include but are not limited to PLA2 from bee venom as well as Apis m1 and m2, Ves g1, g2 and g5, Ves v5 and to Pol and Sol allergens.

Fungal allergens include those from Alternaria alt. and Cladospo. herb. such as Alt a1 and Cla h1.

Food allergens include but are not limited to those from milk (lactoglobulin), egg (ovalbumin), peanuts, hazelnuts, wheat (alpha-amylase inhibitor),

Other plant allergens include latex (hevea brasiliensis).

In addition, a number of proteins of interest for expression in transgenic plants could be useful objects for epitope engineering. If for instance a heterologous enzyme is introduced into a transgenic plant e.g. to increase the nutritional value of food or feed derived from that plant, that enzyme may lead to allergenicity problems in humans or animals ingesting the plant-derived material. Epitope mapping and engineering of such heterologous enzymes or other proteins of transgenic plants may lead to reduction or elimination of this problem. Hence, the methods of this patent are also useful for potentially modifying proteins for heterologous expression in plants and plant cells.

Materials and Methods Materials ELISA Reagents:

Horse Radish Peroxidase labelled pig anti-rabbit-Ig (Dako, DK, P217, dilution 1:1000)
Rat anti-mouse IgE (Serotec MCA419; dilution 1:100)
Mouse anti-rat IgE (Serotec MCA193; dilution 1:200)
Biotin-labelled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140; dilution 1:1000)
Biotin-labelled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; dilution 1:2000)
Streptavidin-horse radish peroxidase (Kirkegård & Perry 14-30-00; dilution 1:1000).

Buffers and Solutions:

PBS (pH 7.2 (1 liter))

NaCl 8.00 g KCl 0.20 g K2HPO4 1.04 g KH2PO4 0.32 g

Washing buffer PBS, 0.05% (v/v) Tween 20

Blocking buffer PBS, 2% (wt/v) Skim Milk powder

Dilution buffer PBS, 0.05% (v/v) Tween 20, 0.5% (wt/v) Skim Milk powder

Citrate buffer 0.1M, pH 5.0-5.2

Stop-solution (DMG-buffer)

Sodium Borate, borax (Sigma)

3,3-Dimethyl glutaric acid (Sigma)

Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184)

PMSF (phenyl methyl sulfonyl flouride) from Sigma

Succinyl-Alanine-Alanine-Proline-Phenylalanine-paranitro-anilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mol.

mPEG (Fluka)

Coloring Substrate:

OPD: o-phenylene-diamine, (Kementec cat no. 4260)

Methods Automatic Epitope Mapping Implementation

The implementation consists of 3 pieces of code:

1. The core program (see above), written in C (see Appendix A).
2. A “wrapping” cgi-script run by the web server, written in Python (see Appendix B).
3. A HTML page defining the input/submission form (see Appendix C).

The wrapper receives the input and calls the core program and several other utilities.

Apart from the standard Unix utility programs (my, rm, awk, etc.) the following must be installed:

A web server capable of running cgi-scripts, e.g. Apache

Python 1.5 or later

Gnuplot 3.7 or later

DSSP, version July 1995

The Core Program Inputs

1. A Brookhaven PDB file with the structure of the protein
2. The output of DSSP called with the above PDB file.
3. Maximum distance between adjacent residues
4. Minimum solvent accessible surface area for each residue
5. Maximum epitope size (max distance between any two residues in epitope)
6. Maximum number of non-redundant epitopes to include (0=all)
7. The shortest acceptable epitope (as a fraction of the length of the epitope consensus sequence).
8. Epitope consensus sequence describing which residues are possible at the different positions. An example is shown below:
KR (Lys or Arg allowed)
AILV− (Ala, Ile, Leu, Val or missing residue allowed)
* (All Residues Allowed, but there Must be a Residue)

? (All or Missing Residue Allowed)

DE (Asp or Glu allowed)
(*, ? or − in first or last position is allowed but obsolete. (− in first position is ignored.))

Examples of matching epitopes:

KAAKD (SEQ ID NO: 41), KLASD (SEQ ID NO: 42), KLYSD (SEQ ID NO: 43), KLY-D (SEQ ID NO: 44), R-M-D. The Epitope Searching Algorithm

The “core” of the program is the algorithm that scans the protein surface for the epitope patterns. The principle is that several “trees” are built, where each of their branches describes one epitope:

1. All residues in the protein are checked according to: a) Does the residue type match the first residue of the epitope consensus sequence. b) Is the surface accessibility greater than or equal to the given threshold. If both requirements are fulfilled, the protein residue is considered as one root in the epitope tree. Remark that there are usually many roots.
2. For each of the residues defined as roots, all residues within the given threshold distance between adjacent residues (e.g. 7 Angstroms) are checked for the same as above: a) Does the residue type match the second residue of the epitope consensus sequence. b) Is the surface accessibility greater than or equal to the given threshold. If yes, the protein residue is considered as a “child” of the root. The spatial position of a residue is defined as the coordinates of its C-alpha atom.
3. The procedure from step 2 is repeated for the next residue in the epitope consensus sequence, where each of the “childs” found in step 2 are now “roots” of new childs. If a gap is defined in the epitope consensus sequence, a “missing” residue is allowed, and the coordinates of the root (also called “parent”) is used.
4. This procedure is repeated for all residues in the epitope consensus sequence.
5. In this way a number of trees (corresponding to the number of roots found in step 1) are found. Notice that the same protein residue can be present many places in the trees.
6. If no epitopes that matches the length of the epitope consensus sequence are found, the longest shorter epitopes that matches the first n residues of the epitope consensus sequence are used, where n is an integer smaller than the length of the epitope consensus sequence. If n is smaller than the length of the epitope consensus sequence multiplied by the fraction value defining the shortest acceptable epitope length, no epitopes are written to the output, and steps 7, 8 and 9 are skipped.
7. The epitopes are extracted from the trees by traversing down from each of the “childs” in the last level. The algorithm also finds epitopes which have the same protein residue present more than once. This is, of course, an artifact and such epitopes are discarded. Every epitope is then checked for its size, that is, the maximum distance between any two residues which are members of the epitope. If this exceeds the threshold, the epitope is discarded.
8. Redundant epitopes are removed. Epitopes containing one or more gaps are redundant if they are subsets of other epitopes without or with fewer gaps. For example: A82-gap-F45-G44-K43 is a subset of A82-L46-F45-G44-K43, and is therefore discarded.
9. For every epitope, the total solvent accessible surface area is calculated (by adding the contributions from each residue as found by the DSSP program). The epitopes are sorted according to this area in descending order. If a maximum number of n non-redundant epitopes has been specified, the n epitopes with largest solvent accessible surface area are selected.
10. The output consists of a list of the found epitopes, along with information of the epitope consensus sequence used and other internal parameters. A separate file containing the number of epitopes that each of the protein residues is a member of is also written.
The wrapper

Inputs

1. One PDB file, describing one structure, or one ZIP file, containing a number of PDB files, each describing one structure. The ZIP file must not contain subfolders.
2. An epitope consensus sequence or which part of the current epitope library to use (full library or IgE part or IgG part).
3. Maximum distance between adjacent residues
4. Minimum solvent accessible surface area for each residue
5. Maximum epitope size (max distance between any two residues in epitope)
6. Maximum number of non-redundant epitopes to include (0=all)
7. Whether to use sequential numbering (1,2,3,4, etc) or PDB-file numbering.

DESCRIPTION

The core program accepts only one structure and one epitope consensus sequence. It is usually desirable to use a library of epitope consensus sequences and sometimes several protein structures. The wrapper reads the user input and calls the utility programs and the core program the necessary number of times. The output is collected and presented on the web page returned to the user.

Depending on the type of input, the wrapper works in different modes:

Epitope consensus can be given directly or taken from a library

Input type can be a single PDB file or a collection of PDB file given as a ZIP-file.

Any of the four possible combinations are allowed.

The epitope library consists of a number of text files, each containing one epitope consensus sequence as specified above.

The layout of the wrapper is like this:

1. Check if the program is already in use from somewhere else (this is done by checking for a lock file when the wrapper starts. If it does not exist, it is created and removed again when the program is finished).
2. If the epitope consensus sequences are to be read from the library, make an internal list of the desired library entries.
3. If the input type is a ZIP file, unzip the file and create one new directory for each of the contained PDB files. Move each PDB file to its corresponding directory.
4. Do a loop over the structures and/or epitope consensus sequences. For each structure/epitope consensus sequence pair, DSSP and the core program is called with the required parameters. If the input type is a ZIP file, the outputs are put in the appropriate directories.
5. If the epitope library is used, a sum file containing the total number of epitopes each residue is a member of. (Such a file is generated by the core program for each epitope consensus sequence—here a sum of these files is calculated). If input type is a ZIP file, a sum file is generated for each structure and put in the appropriate directory.
6. If the epitope library is used, a file containing the total number of epitopes found from each entry in the epitope library. If the input type is a PDB file, the file contains only one line (with a number of data corresponding to the library size). If the input type is a ZIP file, there is one line for each structure.
7. Depending on the combination of input type (ZIP or single PDB) and epitope consensus sequence source (typed-in or epitope library), different information is returned to the user:
Single PDB+typed in epitope: Graph of numbers of epitopes that each residue is a member of. List of found epitopes.
ZIP file+typed in epitope: Graphs (one for each structure) of numbers of epitopes that each residue is a member of. Lists (one for each structure) of found epitopes.
Single PDB+epitope library: Graph of numbers of epitopes that each residue is a member of (total for the complete library).
ZIP file+epitope library: Graphs (one for each structure) of numbers of epitopes that each residue is a member of (total for the complete library).
Data flow sheets for the four different are shown in the FIG.
8. For all modes except Single PDB+typed in epitope, a ZIP file containing all output files is created and returned to the user.

Immunisation of Brown Norway Rats:

Twenty intratracheal (IT) immunisations were performed weekly with 0,100 ml 0.9% (wt/vol) NaCl (control group), or 0,100 ml of a protein dilution (˜0, 1-1 mg/ml). Each group contained 10 rats. Blood samples (2 ml) were collected from the eye one week after every second immunisation. Serum was obtained by blood clothing and centrifugation and analysed as indicated below.

Immunisation of Balb/C Mice:

Twenty subcutaneous (SC) immunisations were performed weekly with 0.05 ml 0.9% (wt/vol) NaCl (control group), or 0.050 ml of a protein dilution (˜0.01-0.1 mg/ml). Each group contained 10 female Balb/C mice (about 20 grams) purchased from Bomholdtgaard, Ry, Denmark. Blood samples (0.100 ml) were collected from the eye one week after every second immunisation. Serum was obtained by blood clothing and centrifugation and analysed as indicated below.

ELISA Procedure for Detecting Serum Levels of IgE and IgG:

Specific IgG1 and IgE levels were determined using the ELISA specific for mouse or rat IgG1 or IgE. Differences between data sets were analysed by using appropriate statistical methods.

Activation of CovaLink Plates:

A fresh stock solution of cyanuric chloride in acetone (10 mg/ml) is diluted into PBS, while stirring, to a final concentration of 1 mg/ml and immediately aliquoted into CovaLink NH2 plates (100 microliter per well) and incubated for 5 minutes at room temperature. After three washes with PBS, the plates are dryed at 50° C. for 30 minutes, sealed with sealing tape, and stored in plastic bags at room temperature for up to 3 weeks.

Mouse anti-Rat IgE was diluted 200× in PBS (5 microgram/ml). 100 microliter was added to each well. The plates were coated overnight at 4° C.

Unspecific adsorption was blocked by incubating each well for 1 hour at room temperature with 200 microliter blocking buffer. The plates were washed 3× with 300 microliter washing buffer.

Unknown rat sera and a known rat IgE solution were diluted in dilution buffer: Typically 10×, 20× and 40× for the unknown sera, and 1/2 dilutions for the standard IgE starting from 1 μg/ml. 100 microliter was added to each well. Incubation was for 1 hour at room temperature.

Unbound material was removed by washing 3× with washing buffer. The anti-rat IgE (biotin) was diluted 2000× in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3× with washing buffer.

Streptavidin was diluted 1000× in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3× with 300 microliter washing buffer. OPD (0.6 mg/ml) and H2O2 (0.4 microliter/ml) were dissolved in citrate buffer. 100 microliter was added to each well. Incubation was for 30 minutes at room temperature. The reaction was stopped by addition of 100 microliter H2SO4. The plates were read at 492 nm with 620 nm as reference.

Similar determination of IgG can be performed using anti Rat-IgG and standard rat IgG reagents.

Similar determinations of IgG and IgE in mouse serum can be performed using the corresponding species-specific reagents.

Direct IgE Assay:

To determine the IgE binding capacity of protein variants one can use an assay, essentially as described above, but using sequential addition of the following reagents:

1) Mouse anti-rat IgE antibodies coated in wells;
2) Known amounts of rat antiserum containing IgE against the parent protein;
3) Dilution series of the protein variant in question (or parent protein as positive control);
4) Rabbit anti-parent antibodies
5) HRPO-labelled anti-rabbit Ig antibodies for detection using OPD as described.

The relative IgE binding capacity (end-point and/or affinity) of the protein variants relative to that of the parent protein are determined from the dilution-response curves. The IgE-positive serum can be of other animals (including humans that inadvertently have been sensitized to the parent protein) provided that the species-specific anti-IgE capture antibodies are changed accordingly.

Competitive ELISA (C-ELISA):

C-ELISA was performed according to established procedures. In short, a 96 well ELISA plate was coated with the parent protein. After proper blocking and washing, the coated antigen was incubated with rabbit anti-enzyme polyclonal antiserum in the presence of various amounts of modified protein (the competitior). The residual amount of rabbit antiserum was detected by horseraddish peroxidase-labelled pig anti-rabbit immunoglobulin.

Protein Sequences and Alignments:

For purposes of the present invention, the degree of homology may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45).

Subtilisin Proteases:

In the present invention, corresponding (or homologous) positions in subtilisin protease sequences are defined by alignment with Subtilisin Novo (BPN′) from B. amyloliquefaciens, as shown in Table 1A for Alcalase, Protease B, Esperase, Protease C, Protease D, Protease E, Protease A, PD498, Properase, Relase, Savinase.

TABLE 1A Alignment of different proteases to the sequence of BPN′ Alcalase: 69.5% identity in 275 residues overlap; Score: 953.0; Gap frequency: 0.4% Alcalase, 1 AQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYN-TD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ** ****   ***     **  * ****** * **  ***** * **** *  *  *  * Alcalase, 60 GNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMD BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ************ *  ********  *******   *** ** *  *****  * ** Alcalase, 120 VINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAV BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV ******** *** * * *** * * ********** ** *   * *** ** ******** Alcalase, 180 DSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPN BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN **   ******** ** *****    ** * * *   *********************** Alcalase, 240 LSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ (SEQ ID NO: 45) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46)     ***  *  * * ** *********** **** Protease B: 59.6% identity in 275 residues overlap; Score: 820.0; Gap frequency: 2.2% PROTEASE B, 1 AQTIPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD **  * * *   *** *  * *** ***** * ** * ****   **** ** * *  ** PROTEASE B, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** ***************  ********  ***  * *  * ***  * * PROTEASE B, 119 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** *  **  *   ** **** ** *         **  *    **** PROTEASE B, 175 DQNNNRASFSQYGAGLDIMAPGVNIQSTYPGSTYASDNGTSMATPHVAGAAALVKQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  *  ** ***** **** **  *   ****** *********   * * PROTEASE B, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 47) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  ** Esperase: 54.7% identity in 274 residues overlap; Score: 745.0; Gap frequency: 2.2% Esperase, 1 QTVPWGISFINTQQAHNRGIFGNGARVAVLDTGI-ASHPDLRIAGGASFISSE-PSYHDN BPN′, 2 QSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQDD * ** * * *     *  *  *    *** * **  *****  *****   ** *   * Esperase, 59 NGHGTHVAGTIAALNNSIGVLGVAPSADLYAVKVLDRNGSGSLASVAQGIEWAINNNMHI BPN′, 62 NSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDV * ******** ***************  *******   ***       ****** *** Esperase, 119 INMSLGSTSGSSTLELAVNRANNAGILLVGAAGNTGRQG----VNYPARYSGVMAVAAVD BPN′, 122 INMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVD ******  ***  *  **  *   *   * **** *  *    * **  *  * ** *** Esperase, 175 QNGQRASFSTYGPEIEISAPGVNVNSTYTGNRYVSLSGTSMATPHVAGVAALVKSRYPSY BPN′, 182 SSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNW    ******  ***    ****   **  ** *    ***** ***** ***  *  * Esperase, 235 TNNQIRQRINQTATYLGSPSLYGNGLVHAGRATQ (SEQ ID NO: 48) BPN′, 242 TNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) ** * *     * * **    ** **     * * Protease C: 59.6% identity in 275 residues overlap; Score: 825.0; Gap frequency: 2.2% ProteaseC, 1 AQSVPWGISRVQAPAAHNRGLTGSGVRVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** * *** * ** * ****   **** ** * *  ** ProteaseC, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSYSSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** ***************  ********  *** ** *  * ***  * * ProteaseC, 119 VASLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV *   *** ** ** *  **  *   ** **** ** *         **  *    **** ProteaseC, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  *  **  ****  *** **  *   ****** *********   * * ProteaseC, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAAAR (SEQ ID NO: 49) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  *** Protease D: 59.3% identity in 275 residues overlap; Score: 815.0; Gap frequency: 2.2% ProteaseD, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** ***** * ** * ****   **** ** * *  ** ProteaseD, 59 GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGAISSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** *** ***********  ********  ***  * *  * ***  * * ProteaseD, 119 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** *  **  *   ** **** ** *         **  *    **** ProteaseD, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  *  **  ****  *** **  *   ****** *********   * * ProteaseD, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 50) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  ** Protease E: 58.2% identity in 275 residues overlap; Score: 800.0; Gap frequency: 2.2% ProteaseE, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** ***** * ** * ****   **** ** * *  ** ProteaseE, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGGGAISSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** ***************  ********  * *  * *  * ***  * * ProteaseE, 119 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----DSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** *  **  *   ** **** ** *         **  *    **** ProteaseE, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKHKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  *  **  ****  *** **  *   ****** ******* *   * * ProteaseE, 235 WSNVRIRDHLKKTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 51) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * *   *  *  * * **    ** ** *  ** Protease A: 58.9% identity in 275 residues overlap; Score: 812.0; Gap frequency: 2.2% Protease A, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** ***** * ** * ****   **** ** * *  ** Protease A, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** ***************  ********  ***  * *  * ***  * * Protease A, 119 VANLSLGSPSAGGTLEQAVNSATSRGVLVVAASGNSGA----GSISAPASYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** **    *  **  *   ** **** ** *          *  *    **** Protease A, 175 DQNNNRASFSQYGPGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  ** **  ****  *** ** *   ****** *********   * * Protease A, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 52) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  ** PD498: 47.7% identity in 266 residues overlap; Score: 487.0; Gap frequency: 4.9% PD498, 13 YGPQNTSTPAAWDVTRGSSTQTVAVLDSGVDYNHPDLARKVIKGYDFIDRDN-NPMDLNG BPN′, 6 YGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDL--KVAGGASMVPSETPNFQDDNS **      **        *   *** *** *  ****  **  *         *  * * PD498, 72 HGTHVAGTVAADTNNGIGVAGMAPDTKILAVRVLDANGSGSLDSIASGIRYAADQGAKVL BPN′, 64 HGTHVAGTVAA-LNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMDVI ***********  ** *** * **     ** **   ***    *  **  *      * PD498, 132 NLSLGCECNSTTLKSAVDYAWNKGAVVVAAAGND----NVSRTFQPASYPNAIAVGAIDS BPN′, 123 NMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAVDS * ***    *  ** *** *   * ********       *    *  **  ***** ** PD498, 188 NDRKASFSNYGTWVDVTAPGVNIASTVPNNGYSYMSGTSMASPHVAGLAALLASQGKN-- BPN′, 183 SNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWT     ****  *   ** **** * ** * * *    *********** ***  *   * PD498, 246 NVQIRQAIEQTADKISGTGTNFKYGK (SEQ ID NO: 53) BPN′, 243 NTQVRSSLQNTTTKL---GDSFYYGK (SEQ ID NO: 46) * * *     *  *    *  * *** Properase: 58.9% identity in 275 residues overlap; Score: 813.0; Gap frequency: 2.2% Properase, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** ***** * ** * ****   **** ** * *  ** Properase, 59 GNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGGGSNSSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** **************   ********  * *  * *  * ***  * * Properase, 119 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** *  **  *   ** **** ** *         **  *    **** Properase, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  *  **  ****  *** **  *   ****** *********   * * Properase, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 54) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  ** Relase: 60.7% identity in 275 residues overlap; Score: 858.0; Gap frequency: 1.8% Relase, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGIDSTHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** ***** * **** ****   **** ** * *  ** Relase, 60 GNGHGTHVAGTIAALDNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMD BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** *** ***********  ********  ***  * *  * ***  * ** Relase, 120 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** *  **  *   ** **** ** *         **  *    **** Relase, 176 DQNNNRASFSQYGAELDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVLQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  * ***  ****  *** **  *   ****** ********* * * * Relase, 236 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 55) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  ** Savinase: 59.6% identity in 275 residues overlap; Score: 821.0; Gap frequency: 2.2% Savinase, 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGE-PSTQD BPN′, 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD ***** * *   *** *  * *** ***** * ** * ****   **** ** * *  ** Savinase, 59 GNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMH BPN′, 61 DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD  * ******** ***************  ********  ***  * *  * ***  * * Savinase, 119 VANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGA----GSISYPARYANAMAVGAT BPN′, 121 VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * * *** ** ** *  **  *   ** **** ** *         **  *    **** Savinase, 175 DQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPS BPN′, 181 DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *  * *****  *  **  ****  *** **  *   ****** *********   * * Savinase, 235 WSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR (SEQ ID NO: 56) BPN′, 241 WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) * * * *  * ** * **    ** ** *  **

To find the homologous positions in subtilisin protease sequences not shown in the alignment of Table 1A, the sequence of interest is aligned to the sequence of BPN′ as shown in Table 1B for YaB protease and Subtilisin sendai. The new sequence is aligned to the BPN′ sequence by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45).

The sequence of the YaB protease is disclosed by Kaneko, R.; Koyama, N.; Tsai, Y.-C.; Juang, R.-Y.; Yoda, K.; Yamasaki, M.; Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain. J. Bacteriol. 171:5232 (1989), it has Swissprot number P20724, and is shown in SEQ ID NO: 35.

The sequence of the Subtilisin sendai is disclosed by Yamagata, Y.; Isshiki, K.; Ichishima, E.; Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS. Enzyme Microb. Technol. 17:653 (1995), it has SPTREMBL accession number Q45522, and is shown in SEQ ID NO: 34.

Identity to savinase: 81.7%
identity to savinase: 82.09%

Swissprot: P20724

TABLE 1B Alignment of YAB protease to BPN′: 55.3% identity CLUSTAL W (1.7) multiple sequence alignment YAB -QTVPWGINRVQAPIAQSRGFTGTGVRVAVLDTGISN-HADLRIRGGASFVPGE-PNISD BPN{acute over ( )} AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD  *:**:*:.:::**  :*:*:**:.*:***:*:**.. *.**:: ****:**.* **:.* YAB GNGHGTQVAGTIAALNNSIGVLGVAPNVDLYGVKVLGASGSGSISGIAQGLQWAANNGMH BPN{acute over ( )} DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD .*.***:****:**************.  **.***** :***. * * :*::**  *.*. YAB IANMSLGSSAGSATMEQAVNQATASGVLVVAASGNSG----AGNVGFPARYANAMAVGAT BPN{acute over ( )} VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV : *****..:***::: **::*.****:****:**.*    :..**:*.:*...:****. YAB DQNNNRATFSQYGAGLDIVAPGVGVQSTVPGNGYASFNGTSMATPHVAGVAALVKQKNPS BPN{acute over ( )} DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *..*:**:**. *. **::****.:***:*** *.::******:*****.***: .*:*. YAB WSNVQIRNHLKNTATNLGNTTQFGSGLVNAEAATR (SEQ ID NO: 57) BPN{acute over ( )} WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) *:*.*:*. *:**:*:**::  :*.**:*.:**:: Alignment of Subtilisin sendai to BPN′: 55.6% identity. CLUSTAL W (1.7) multiple sequence alignment sendai NQVTPWGITRVQAPTAWTRGYTGTGVRVAVLDTGIS-THPDLNIRGGVSFVPGE-PSYQD BPN{acute over ( )} AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETPNFQD  * .*:*:::::**:  ::****:.*:***:*:**. :****:: **.*:**.* *.:** sendai GNGHGTHVAGTIAALNNSIGVVGVAPNAELYAVKVLGANGSGSVSSIAQGLQWTAQNNIH BPN{acute over ( )} DNSHGTHVAGTVAALNNSIGVLGVAPSSALYAVKVLGDAGSGQYSWIINGIEWAIANNMD .*.********:*********:****.: ********  ***. * * :*::*:  **:. sendai VANLSLGSPVGSQTLELAVNQATNAGVLVVAATGNNG----SGTVSYPARYANALAVGAT BPN{acute over ( )} VINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGSTGSSSTVGYPGKYPSVIAVGAV * *:***.* ** :*: **::*. :**:****:**:*    *.**.**.:*...:****. sendai DQNNNRASFSQYGTGLNIVAPGVGIQSTYPGNRYASLSGTSMATPHVAGVAALVKQKNPS BPN{acute over ( )} DSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPN *..*:*****. *. *:::****.**** ***:*.: .*****:*****.***: .*:*. sendai WSNTQIRQHLTSTATSLGNSNQFGSGLVNAEAATR (SEQ ID NO: 58) BPN{acute over ( )} WTNTQVRSSLQNTTTKLGDSFYYGKGLINVQAAAQ (SEQ ID NO: 46) *:***:*. * .*:*.**:*  :*.**:*.:**::

These alignments reveal that that homology between various subtilisin proteases ranges between 100% and 40%.

Unless specified, subtilisin sequences and positions mentioned in the present invention, are given in the BPN′ numeration, and can be converted by alignment as 50 described above (Tables 1A and 1B).

Sequence identities between different pairs of proteases are given below:

Sequence identity to BPN′:

Savinase 60.4% Alcalase 69.5% BLAPR 60.4% ProteaseC 0.4% ProteaseD 0.0% ProteaseE 8.2% Protease A 0.0% Properase 9.6% Relase 61.5% PD498 44.8% sendai 55.6% YAB 55.3%

Sequence identity to Savinase:

Alcalase 60.9% BLAPR 98.1% ProteaseC 8.5% ProteaseD 8.9% ProteaseE 6.7% Protease A 7.8% Properase 8.9% Relase 98.1% PD498 44.3% sendai 81.4% YAB 81.8%

Structures

The protein structure of PD498 is disclosed in WO 98/35026 (Novo Nordisk). The structure of Savinase can be found in BETZEL et al, J.MOL.BIOL., Vol. 223, p. 427, 1992 (1 svn.pdb).

Homology Modelling

Three dimensional structural models of the subtilisins properase, release, ProteaseC, ProteaseD, ProteaseE, and PROTEASE B were constructed based on three dimensional structure of Savinase (Protein Data Bank entry 1SVN; Betzel, C., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S., Wilson, K. S.: Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 Å resolution. J Mol Biol 223 pp. 427 (1992)) using the Modeller 5o ( Sali, A.; T. L. Blundell, “Definition of general topological equivalence in protein structures: A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming,” J. Mol. Biol., 212 403-428 (1990)) module of the Insight 2000 molecular modelling package (Biosym inc.). Default parameters were used with the alignments shown in FIG. 1A as input, e.g. alignment between the columns labelled Savinase and PROTEASE B served as input alignment in construction of a PROTEASE B structural model. The Modeller module by default output ten structural models, of these the model with lowest ‘modeller objective function’ score was chosen as representing PROTEASE B structure.

Lipase:

The sequence of the T. lanuginosus lipase (trade name Lipolase) is provided in SEQ ID NO: 1 and the structure is disclosed in WO 98/35026 and as “1tib”, available in Structural Classification of Proteins (SCOP) on the Internet.

Amylase:

The amylase used in the examples is the alpha-amylase of Bacillus halmapalus (WO 96/23873), which is called amylase SP722 (the wild-type). Its sequence is shown in SEQ ID NO: 2 and the corresponding protein structure was built from the BA2 structure, as described in WO 96/23874. The first four amino acids of the structural model are not defined, hence the sequence used for numeration of amino acid residues in the examples of this invention is four amino acids shorter than the one of the full length protein SP722.

Several variants of this amylase are available (WO 96/23873). One particularly useful variant has deleted two amino acid residues at D-G at positions 183 and 184 of the SEQ ID NO: 2 (corresponding to residues 179 and 180 of the modelled structure). This variant is called JE-1 or Natalase.

Another amylase that is particularly useful is the amylase AA560: This alkaline alpha-amylase may be derived from a strain of Bacillus sp. DSM 12649. The strain was deposited on 25 Jan. 1999 by the assignee under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE.

Laccase:

The laccase used in this invention is that from Coprinus cinereus (WO 98/38287), the sequence of which is shown as SEQ ID NO: 3. The structure of the Myceliophthora thermophila laccase can be built by homology modeling to the Coprinus cinereus laccase as shown in WO 98/38287.

Cellulase:

The cellulase sequence and structure used in the present invention is that of the core fragment of endoglucanase V from Humicola insolens (aka Cel45 or Carezyme). The core fragment structure is available as 3eng.pdb (G. J. DAVIES et al. ACTA CRYSTALLOGR., SECT.D, Vol. 52, p. 7 1996; G. J. DAVIES et al. BIOCHEMISTRY, V. 34, p. 16210, 1995); SwissProt accession number P43316, and the sequences shown in SEQ ID 4. The corresponding full-length sequence is disclosed in WO 91/17243 and shown here in SEQ ID NO: 5. The numeration of all description and claims of this invention pertain to the core fragment, however, it is contemplated that all claims are also valid for the corresponding positions in the full-length protein.

TABLE 1 Alignment and numeration scheme for subtilisins (SEQ ID NOS: 46, 45, 47, 48, 49, 50, 51, 52, 54, 55, 56, 53, respectively) Pro- Pro- Pro- Pro- Pro- BPN′ Alcalase teaseB Esperase teaseC teaseD teaseE teaseA Properase Relase Savinase PD498  −6 W  −5 S  −4 P  −3 N  −2 D  −1 P   1 A A A A A A A A A A Y   2 Q Q Q Q Q Q Q Q Q Q Q Y   3 S T T T S S S S S S S S   3a A   4 V V I V V V V V V V V Y   5 P P P P P P P P P P P Q   6 Y Y W W W W W W W W W Y   7 G G G G G G G G G G G G   8 V I I I I I I I I I I P   9 S P S S S S S S S S S Q  10 Q L R F R R R R R R R N  11 I I V I V V V V V V V T  12 K K Q N Q Q Q Q Q Q Q S  13 A A A T A A A A A A A T  14 P D P Q P P P P P P P P  15 A K A Q A A A A A A A A  16 L V A A A A A A A A A A  17 H Q H H H H H H H H H W  18 S A N N N N N N N N N D  19 Q Q R R R R R R R R R V  20 G G G G G G G G G G G  21 Y F L I L L L L L L L T  22 T K T F T T T T T T T R  23 G G G G G G G G G G G G  24 S A S N S S S S S S S S  25 N N G G G G G G G G G S  26 V V V A V V V V V V V T  27 K K K R R K K K K K K Q  28 V V V V V V V V V V V T  28a V  29 A A A A A A A A A A A A  30 V V V V V V V V V V V V  31 I L L L L L L L L L L L  32 D D D D D D D D D D D D  33 S T T T T T T T T T T S  34 G G G G G G G G G G G G  35 I I I I I I I I I I I V  36 D Q D D  37 S A S A S S S S S S S Y  38 S S T S T T T T T T T N  39 H H H H H H H H H H H H  40 P P P P P P P P P P P P  41 D D D D D D D D D D D D  42 L L L L L L L L L L L L  43 K N N R N N N N N N N A  44 V V I I I I I I I I I R  44a K  44b V  45 A V R A R R R R R R R I  46 G G G G G G G G G G G K  47 G G G G G G G G G G G G  48 A A A A A A A A A A A Y  49 S S S S S S S S S S S D  50 M F F F F F F F F F F F  51 V V V I V V V V V V V I  52 P A P S P P P P P P P D  53 S G G S G G G G G G G R  54 E E E E E E E E E E E D  55 T A P P P P P P P P N  56 P Y P N  57 N N S S S S S S S S S P  58 F T Y T T T T T T T M  59 Q T Q H Q Q Q Q Q Q Q  60 D D D D D D D D D D D D  61 D G G N G G G G G G G L  62 N N N N N N N N N N N N  63 S G G G G G G G G G G G  64 H H H H H H H H H H H H  65 G G G G G G G G G G G G  66 T T T T T T T T T T T T  67 H H H H H H H H H H H H  68 V V V V V V V V V V V V  69 A A A A A A A A A A A A  70 G G G G G G G G G G G G  71 T T T T T T T T T T T T  72 V V I I I I I I I I I V  73 A A A A A A A A A A A A  74 A A A A A A A A A A A A  75 L L L L L L L L L L L D  75a T  76 N D N N N D N N N D N N  77 N N N N N N N N N N N N  78 S T S S S S S S S S S G  79 I T I I I I I I I I I I  80 G G G G G G G G G G G G  81 V V V V V V V V V V V V  82 L L L L L L L L L L L A  83 G G G G G G G G G G G G  84 V V V V V V V V V V V M  85 A A A A A A A A A A A A  86 P P P P P P P P P P P P  87 S S S S S S S S N S S D  88 S V A A A A A A A A A T  89 A S E D E E E E E E E K  90 L L L L L L L L L L L I  91 Y Y Y Y Y Y Y Y Y Y Y L  92 A A A A A A A A A A A A  93 V V V V V V V V V V V V  94 K K K K K K K K K K K R  95 V V V V V V V V V V V V  96 L L L L L L L L L L L L  97 G N G D G G G G G G G D  98 D S A R A A A A A A A A  99 A S S N S S S S S S S N 100 G G G G G G G G G G G G 101 S S S S S S G S G S S S 102 G G G G G G G G G G G G 103 Q S S S S A A S S S S S 104 Y Y V L Y I I V N V V L 105 S S S A S S S S S S S D 106 W G S S S S S S S S S S 107 I I I V I I I I I I I I 108 I V A A A A A A A A A A 109 N S Q Q Q Q Q Q Q Q Q S 110 G G G G G G G G G G G G 111 I I L I L L L L L L L I 112 E E E E E E E E E E E R 113 W W W W W W W W W W W Y 114 A A A A A A A A A A A A 115 I T G I G G G G G G G A 116 A T N N N N N N N N N D 117 N N N N N N N N N N N Q 118 N G G N G G G G G G G G 119 M M M M M M M M M M M A 120 D D H H H H H H H D H K 121 V V V I V V V V V V V V 122 I I A I A A A A A A A L 123 N N N N S N N N N N N N 124 M M L M L L L L L L L L 125 S S S S S S S S S S S S 126 L L L L L L L L L L L L 127 G G G G G G G G G G G G 128 G G S S S S S S S S S C 129 P A P T P P P P P P P E 130 S S S S S S S S S S S C 131 G G P G P P P A P P P N 132 S S S S S S S G S S S S 133 A T A S A A A G A A A T 134 A A T T T T T T T T T T 135 L M L L L L L L L L L L 136 K K E E E E E E E E E K 137 A Q Q L Q Q Q Q Q Q Q S 138 A A A A A A A A A A A A 139 V V V V V V V V V V V V 140 D D N N N N N N N N N D 141 K N S R S S S S S S S Y 142 A A A A A A A A A A A A 143 V Y T N T T T T T T T W 144 A A S N S S S S S S S N 145 S R R A R R R R R R R K 146 G G G G G G G G G G G G 147 V V V I V V V V V V V A 148 V V L L L L L L L L L V 149 V V V L V V V V V V V V 150 V V V V V V V V V V V V 151 A A A G A A A A A A A A 152 A A A A A A A A A A A A 153 A A S A S S S S S S S A 154 G G G G G G G G G G G G 155 N N N N N N N N N N N N 156 E S S T S S S S S S S D 157 G G G G G G G G G G G N 158 S S A R A A A A A A A V 159 T S Q 160 G G G G G G D G G G G S 161 S N S S S S S S S S R 162 S T I I I I I I I I T 163 S N S S S S S S S S F 164 T T 165 V I V 166 G G N 167 Y Y Y Y Y Y Y A Y Y Y Q 168 P P P P P P P P P P P P 169 G A A A A A A A A A A A 170 K K R R R R R S R R R S 171 Y Y Y Y Y Y Y Y Y Y Y Y 172 P D A S A A A A A A A P 173 S S N G N N N N N N N N 174 V V A V A A A A A A A A 175 I I M M M M M M M M M I 176 A A A A A A A A A A A A 177 V V V V V V V V V V V V 178 G G G A G G G G G G G G 179 A A A A A A A A A A A A 180 V V T V T T T T T T T I 181 D D D D D D D D D D D D 182 S S Q Q Q Q Q Q Q Q Q S 183 S N N N N N N N N N N N 184 N S N G N N N N N N N D 185 Q N N Q N N N N N N N R 186 R R R R R R R R R R R K 187 A A A A A A A A A A A A 188 S S S S S S S S S S S S 189 F F F F F F F F F F F F 190 S S S S S S S S S S S S 191 S S Q T Q Q Q Q Q Q Q N 192 V V Y Y Y Y Y Y Y Y Y Y 193 G G G G G G G G G G G G 194 P A A P A A A P A A A T 195 E E G E G G G G G E G W 196 L L L I L L L L L L L V 197 D E D E D D D D D D D D 198 V V I I I I I I I I I V 199 M M M S V V V V V V V T 200 A A A A A A A A A A A A 201 P P P P P P P P P P P P 202 G G G G G G G G G G G G 203 V A V V V V V V V V V V 204 S G N N N N N N N N N N 205 I V I V V V V V V V V I 206 Q Y Q N Q Q Q Q Q Q Q A 207 S S S S S S S S S S S S 208 T T T T T T T T T T T T 209 L Y Y Y Y Y Y Y Y Y Y V 210 P P P T P P P P P P P P 211 G T G G G G G G G G G N 212 N N S N S S S S S S S N 213 K T T R T T T T T T T G 214 Y Y Y Y Y Y Y Y Y Y Y Y 215 G A A V A A A A A A A S 216 A T S S S S S S S S S Y 217 Y L D L L L L L L L L M 218 N N N S N N N N N N N S 219 G G G G G G G G G G G G 220 T T T T T T T T T T T T 221 S S S S S S S S S S S S 222 M M M M M M M M M M M M 223 A A A A A A A A A A A A 224 S S T T T T T T T T T S 225 P P P P P P P P P P P P 226 H H H H H H H H H H H H 227 V V V V V V V V V V V V 228 A A A A A A A A A A A A 229 G G G G G G G G G G G G 230 A A A V A A A A A A A L 231 A A A A A A A A A A A A 232 A A A A A A V A A A A A 233 L L L L L L L L L L L L 234 I I V V V V V V V V V L 235 L L K K K K K K K L K A 236 S S Q S Q Q H Q Q Q Q S 237 K K K R K K K K K K K Q 238 H H N Y N N N N N N N G 239 P P P P P P P P P P P K 240 N N S S S S S S S S S N 241 W L W Y W W W W W W W 242 T S S T S S S S S S S 243 N A N N N N N N N N N N 244 T S V N V V V V V V V V 245 Q Q Q Q Q Q R Q Q Q Q Q 246 V V I I I I I I I I I I 247 R R R R R R R R R R R R 248 S N N Q N N D N N N N Q 249 S R H R H H H H H H H A 250 L L L I L L L L L L L I 251 Q S K N K K K K K K K E 252 N S N Q N N K N N N N Q 253 T T T T T T T T T T T T 254 T A A A A A A A A A A A 255 T T T T T T T T T T T D 256 K Y S Y S S S S S S S K 257 L L L L L L L L L L L I 258 G G G G G G G G G G G S 259 D S S S S S S S S S S G 260 S S T P T T T T T T T T 261 F F N S N N N N N N N G 262 Y Y L L L L L L L L L T 263 Y Y Y Y Y Y Y Y Y Y Y N 264 G G G G G G G G G G G F 264a K 265 K K S N S S S S S S S Y 266 G G G G G G G G G G G G 267 L L L L L L L L L L L K 268 I I V V V V V V V V V I 269 N N N H N N N N N N N N 270 V V A A A A A A A A A S 271 Q E E G E E E E E E E N 272 A A A R A A A A A A A K 273 A A A A A A A A A A A A 274 A A T T A T T T T T T V 275 Q Q R Q R R R R R R R R 276 Y

EXAMPLES Example 1 Identification of Epitope Sequences and Epitope Patterns

High diversity libraries (1012) of phages expressing random hexa-, nona- or dodecapetides as part of their membrane proteins, were screened for their capacity to bind purified specific rabbit IgG, and purified rat and mouse IgG1 and IgE antibodies. The phage libraries were obtained according to prior art (se WO 9215679 hereby incorporated by reference).

The antibodies were raised in the respective animals by subcutaneous, intradermal, or intratracheal injection of relevant proteins (e.g. proteases, lipolytic enzymes, amylases, oxidoreductases) dissolved in phosphate buffered saline (PBS). The respective antibodies were purified from the serum of immunised animals by affinity chromatography using paramagnetic immunobeads (Dynal AS) loaded with pig anti-rabbit IgG, mouse anti-rat IgG1 or IgE, or rat anti-mouse IgG1 or IgE antibodies.

The respective phage libraries were incubated with the IgG, IgG1 and IgE antibody coated beads. Phages, which express oligopeptides with affinity for rabbit IgG, or rat or mouse IgG1 or IgE antibodies, were collected by exposing these paramagnetic beads to a magnetic field. The collected phages were eluted from the immobilised antibodies by mild acid treatment, or by elution with intact enzyme. The isolated phages were amplified as know to the specialist. Alternatively, immobilised phages were directly incubated with E. coli for infection. In short, F-factor positive E. coli (e.g. XL-1 Blue, JM101, TG1) were infected with M13-derived vector in the presence of a helper-phage (e.g. M13K07), and incubated, typically in 2xYT containing glucose or IPTG, and appropriate antibiotics for selection. Finally, cells were removed by centrifugation. This cycle of events was repeated 2-5 times on the respective cell supernatants. After selection round 2, 3, 4, and 5, a fraction of the infected E. coli was incubated on selective 2xYT agar plates, and the specificity of the emerging phages was assessed immunologically. Thus, phages were transferred to a nitrocellulase (NC) membrane. For each plate, 2 NC-replicas were made. One replica was incubated with the selection antibodies, the other replica was incubated with the selection antibodies and the immunogen used to obtain the antibodies as competitor. Those plaques that were absent in the presence of immunogen, were considered specific, and were amplified according to the procedure described above.

The specific phage-clones were isolated from the cell supernatant by centrifugation in the presence of polyethylenglycol. DNA was isolated, the DNA sequence coding for the oligopeptide was amplified by PCR, and the DNA sequence was determined, all according to standard procedures. The amino acid sequence of the corresponding oligopeptide was deduced from the DNA sequence.

Thus, a number of peptide sequences with specificity for the protein specific antibodies, described above, were obtained. These sequences were collected in a database, and analysed by sequence alignment to identify epitope patterns. For this sequence alignment, conservative substitutions (e.g. aspartate for glutamate, lysine for arginine, serine for threonine) were considered as one. This showed that most sequences were specific for the protein the antibodies were raised against. However, several cross-reacting sequences were obtained from phages that went through 2 selection rounds only. In the first round 22 epitope patterns were identified.

In further rounds of phage display, more antibody binding sequences were obtained leading to more epitope patterns. Further, the literature was searched for peptide sequences that have been found to bind environmental allergen-specific antibodies (J All Clin Immunol 93 (1994) pp. 34-43; Int Arch Appl Immunol 103 (1994) pp. 357-364; Clin Exp Allergy 24 (1994) pp. 250-256; Mol Immunol 29 (1992) pp. 1383-1389; J Immunol 121 (1989) pp. 275-280; J. Immunol 147 (1991) pp. 205-211; Mol Immunol 29 (1992) pp. 739-749; Mol Immunol 30 (1993) pp. 1511-1518; Mol Immunol 28 (1991) pp. 1225-1232; J. Immunol 151 (1993) pp. 7206-7213). These antibody binding peptide sequences were included in the database.

A first generation database of antibody binding peptides identified and their corresponding epitope patterns are shown in Table 2-7 below.

Tables 2-7: Overview of the antibody binding peptide sequences, epitope patterns and epitope sequences. The type of antibody used for identifying the antibody binding sequences is indicated as IgG or IgE and the species from which the antibodies were derived are indicated as mo (mouse), ra (rat) and hu (human).

TABLE 2 Savinase antibody binding peptide sequences, epitope patterns and epitope sequences. Antibody binding Method of Epitope peptide identification pattern Donor Acceptor Epitope Sequence (BPN') VQVYGDTSA (SEQ Phage Q > Y > D > Savinase savinase Q206 V81 Y214 G80 D41 ID NO: 59) display T208 LQCVGS (SEQ ID Protein a-amylase savinase L21 Q236 V26 G25 S24 NO: 60) fragments inhibitor KRFANTELA (SEQ Phage R/K R F > N Savinase savinase K251 R247 A174 N173 ID NO: 61) display LDQIFFTRW (SEQ Phage D/E Q I F F T Savinase savinase L42/L75 D41 Q2 I79 ID NO: 62) display FNDAFFVKM (SEQ Phage Savinase savinase N185 D181 A187 F189 ID NO: 63) display V203 ANIPIWSRSA (SEQ Phage > R S A Savinase savinase R145 S144 A142 ID NO: 64) display ANIPIWSRSA (SEQ Phage > R S A Savinase savinase S188 R186 S190 A179 ID NO: 64) display RQSTDFGTT (SEQ Phage R Q > > Savinase savinase R186 Q191 S156 ID NO: 65) display D/E VQVYGDTSA (SEQ Phage Q > Y > D > Savinase savinase Q191 Y192 ID NO: 66) display G193/A194/G195 D197 S265 RRFSNATRA (SEQ Phage R/K R F > N Savinase savinase K251 R247 A174 N173 ID NO: 67) display CTARLRAGNACG Phage A R > A Savinase savinase A172/A169 R170 A194 (SEQ ID NO: 68) display G193 N261 LDQIFFTRW (SEQ Phage D/E Q I F F T Savinase savinase D60 Q59 I44/I35 ID NO: 69) display LDQIFFTRW (SEQ Phage D/E Q I F F T Savinase savinase L42/L75 D41 Q2 I79 ID NO: 69) display EQIFFTSGL (SEQ ID Phage D/E Q I F F T Savinase savinase E112 Q109 I79 NO: 70) display GRFSNSKFK (SEQ Phage L > G R S Savinase savinase L196 G195 R170 S163 ID NO: 71) display AVLRDC (SEQ ID Protein a-amylase savinase A254 V268 L267 R10 D181 NO: 72) fragments inhibitor LQCVGS (SEQ ID Protein a-amylase savinase L217 Q206 V81 G80 S3 NO: 73) fragments inhibitor LRQCNERCV (SEQ Phage R Q > > Savinase savinase L267 R10 Q12 N269 E271 ID NO: 74) display D/E R275 SPVTKRASLKIDSKK Protein Der p II savinase A88 S87/T22 L233 K235 (SEQ ID NO: 75) fragments I246 RQSTDFGTT (SEQ Phage R Q > > Savinase savinase R247 Q245 S240/S242 ID NO: 76) display D/E FCTNNCELS (SEQ Phage N > > E L Savinase savinase T143 N173 N140 E136 L135 ID NO: 77) display FCTNNCELS (SEQ Phage N > > E L Savinase savinase N117 N116 E112 L111 ID NO: 77) display DFHVKYAAQ (SEQ Phage Savinase savinase ID NO: 78) display VAQYKALPVVLENA Protein Fel d I savinase L135 P168 V139 L111 E112 (SEQ ID NO: 79) fragments N116 AAYPDV (SEQ ID Protein A > > > > Y a-amylase savinase A215 Y214 P40 D41 V81 NO: 80) fragments P > inhibitor EQIFFTSGL (SEQ ID Phage D/E Q I F F T Savinase savinase E271 Q12 I8 NO: 81) display VDAAF (SEQ ID NO: Protein Poa p IX savinase V203 D181 A179 A187 F189 82) fragments AVLRDC (SEQ ID Protein a-amylase savinase A232 V234 L250 R247 D197 NO: 83) fragments inhibitor RAFRRNANW (SEQ Phage A R > A Savinase savinase A272/A273 R275 R19 N18 ID NO: 84) display A15/A16 CTARLRAGNACG Phage A R > A Savinase savinase A15/A16 R19 L21 R275 (SEQ ID NO: 85) display A272 A273 N269 TFHDAPALQ (SEQ Phage Savinase savinase H39 D41 A74/A73 P86 A88 ID NO: 86) display L90 CTARVVALGVCG Phage A R > A Savinase savinase R145 V147 V149 A151 (SEQ ID NO: 87) display L124/L126 G127 GRFSNSKFK (SEQ Phage L > G R S Savinase savinase L148 G146 R145 ID NO: 88) display S144/S141 N140 RRFANDHTR (SEQ Phage R/K R F > N savinase savinase K27 R45 N43 D41 H39 ID NO: 89) display T38/T213 KRFANTEPA (SEQ Phage R/K R F > N savinase savinase K251 R247 A174 N173 ID NO: 90) display YKVSAL (SEQ ID Protein a-amylase savinase Y91 K27 V26 S24 G23 L21 NO: 91) fragments inhibitor TGKYVS (SEQ ID Protein a-amylase savinase S24 G25 K27 Y91 V93 NO: 92) fragments inhibitor Antibody binding peptide Epitope # IgG IgE VQVYGDTSA (SEQ sav1.1 Ra ID NO: 59) LQCVGS (SEQ ID sav19.1 Hu NO: 60) KRFANTELA (SEQ sav6.1 Ra- Mo ID NO: 61) Mo LDQIFFTRW (SEQ sav5.1 Ra ID NO: 62) FNDAFFVKM (SEQ sav11.0 Ra ID NO: 63) ANIPIWSRSA (SEQ sav3.2-lac1.0- Ra ID NO: 64) lip4.0-pd5.0 ANIPIWSRSA (SEQ sav3.1-lac1.0- Ra ID NO: 64) lip4.0-pd5.0 RQSTDFGTT (SEQ sav2.2 Ra ID NO: 65) VQVYGDTSA (SEQ sav1.2 Ra ID NO: 66) RRFSNATRA (SEQ sav6.1 Ra- Mo ID NO: 67) Mo CTARLRAGNACG sav10.4 Ra (SEQ ID NO: 68) LDQIFFTRW (SEQ sav5.2 Ra ID NO: 69) LDQIFFTRW (SEQ sav5.1 Ra ID NO: 69) EQIFFTSGL (SEQ ID sav5.4 Ra NO: 70) GRFSNSKFK (SEQ sav9.2-je4.0-lip5.1-5.2 Ra ID NO: 71) AVLRDC (SEQ ID sav18.1-pd18.1-18.2 Hu NO: 72) LQCVGS (SEQ ID sav19.2 Hu NO: 73) LRQCNERCV (SEQ sav2.1 Ra ID NO: 74) SPVTKRASLKIDSKK sav16.0-pd7.0 Hu (SEQ ID NO: 75) RQSTDFGTT (SEQ sav2.3 Ra ID NO: 76) FCTNNCELS (SEQ sav7.2 Ra ID NO: 77) FCTNNCELS (SEQ sav7.1 Ra ID NO: 77) DFHVKYAAQ (SEQ sav8.0 Ra ID NO: 78) VAQYKALPVVLENA sav12.0-pd8.0 Hu (SEQ ID NO: 79) AAYPDV (SEQ ID sav13.0-pd13.1-13.2 Hu NO: 80) EQIFFTSGL (SEQ ID sav5.3 Ra NO: 81) VDAAF (SEQ ID NO: sav15.0-pd12.0 Hu 82) AVLRDC (SEQ ID sav18.2-pd18.1-18.2 Hu NO: 83) RAFRRNANW (SEQ sav10.1 Ra ID NO: 84) CTARLRAGNACG sav10.2 Ra (SEQ ID NO: 85) TFHDAPALQ (SEQ sav4.0 Ra ID NO: 86) CTARVVALGVCG sav10.3 Ra (SEQ ID NO: 87) GRFSNSKFK (SEQ sav9.1-je4.0-lip5.1-5.2 Ra ID NO: 88) RRFANDHTR (SEQ sav6.2 Ra ID NO: 89) KRFANTEPA (SEQ sav6.1 Ra- Mo ID NO: 90) Mo YKVSAL (SEQ ID sav14.0-pd14.0 Hu NO: 91) TGKYVS (SEQ ID sav17.0-pd17.1-17.2 Hu NO: 92)

TABLE 3 PD498 antibody binding peptide sequences, epitope patterns and epitope sequences. Epitope pattern Donor Acceptor Epitope Sequence (BPN′) Epitope # IgG IgE Fel d I pd498 V198 A254 Q252 Y276 K239 A235 L233 P86 pd8.0 Hu A > > > a-amylase pd498 *3aA Y1/Y2 P-4/P-1 D-2 V81 pd13.2 Hu > Y P > inhibitor Poa p IX pd498 S182 Y6 G7 P8 T13 P14 A15 A16 pd11.0 Hu > K L > > Poa p IX pd498 Y171 K136 L135 A108 Y113 pd4.4 Hu a-amylase pd498 Y48/Y37 K46 *44aaV A43 L42 pd14.0 Hu inhibitor Poa p IX pd498 V196/V198 D197 A174/A176 A169 F163 pd12.0 Hu K Q S Poa p IX pd498 A142 A147 V148 K120 Q27 S24/S25 pd2.3 Hu K Q S pd498 pd498 R44 K89 Q27 S236 K120 G146 pd2.2 Ra Der p II pd498 *28aV T88 *44a K R44 A43 L42 pd7.0 Hu > K L > > pd498 pd498 N56/N55 K46 L91 A29/A119 T28 pd4.3 Ra > K L > > pd498 pd498 N240/N243 K239 L233/L234 A16 T21 R22 pd4.1 Ra > K L > > Poa p IX pd498 Y37 K46 L91 A114 Y113 pd4.5 Hu > K L > > pd498 pd498 N240/N243 K239 L233/L234 A16 T21 R22 pd4.1 Ra Y I > K L pd498 pd498 Y113 I111 A108/A138 K136 L135 pd3.1 Ra K Q S pd498 pd498 A115 K145 N243 N240 K239 Q237 S236 pd2.1 Ra > R Y > K/R pd498 pd498 R94 R53 Y48 Q117 R112 S109/S137 pd1.5-lac2.0 Ra Phl p V pd498 A169 Q167 F163 T162 S160 G193 pd10.0 Hu Y I > K L pd498 pd498 Y276 I246 K239 L234 S236 pd3.2 Ra > K L > > pd498 pd498 N240/N243 K239 L233/L234 R22 P86 pd4.2 Ra A > > > a-amylase pd498 *3aA Y2 P14 D18 V19 pd13.1 Hu > Y P > inhibitor K Q S Poa p IX pd498 A15 A16 V274 K239 Q237 S236 pd2.4 Hu a-amylase pd498 G146 K145 Y141 V139 S137 pd17.2 Hu inhibitor a-amylase pd498 A273 V274 L233 R22 D87 pd18.1 Hu inhibitor A R > A Par j 1 + pd498 N10 S12 A15/A16 R275 A273/A249 R247 pd9.0 Hu + Ra Hu Par o 1 A174 D197 S170 pd498 pd498 R22 G23 L233 S236 pd6.2 Ra > R Y > K/R pd498 pd498 R94 R53 Y48 P57 K46 L91 pd1.4-lac2.0 Ra > R Y > K/R pd498 pd498 R94 R53 Y48 P57 K46 L91 pd1.4-lac2.0 Ra a-amylase pd498 L96 R94 S33 V35 Y37 pd15.0 Hu inhibitor > R Y > K/R pd498 pd498 S109/S137 R112 Y141 N144 K145 pd1.3-lac2.0 Ra > R Y > K/R pd498 pd498 T162 R161 Y192 N191 K186 pd1.2-lac2.0 Ra > R Y > K/R pd498 pd498 T133/T134 R112 Y141 N144 K145 pd1.1-lac2.0 Ra a-amylase pd498 A92 *44aaV L42 R44 D75 pd18.2 Hu inhibitor a-amylase pd498 S236 G238 K239 Y276 V274 S270 pd17.1 Hu inhibitor a-amylase pd498 S12 P14 W17 S-5 W-6 pd16.0 Hu inhibitor > R S A pd498 pd498 S137 R112 S109 A108 pd5.0-lac1.0-lip4.0- Ra sav3.1-3.2 pd498 pd498 S215 M217 I205 M222 G219 pd6.1 Ra

TABLE 4 Antibody binding peptide sequences, epitope patterns and epitope sequences for the T. lanuginosus lipase (Lipolase). Antibody binding Method of Epitope peptide identification pattern Donor Acceptor Epitope Sequence Epitope # IgG IgE QRPPRYELE (SEQ Phage R P P R lipolase lipolase lip1.0 Ra ID NO: 93) display ELEYRPPRQ (SEQ Phage > E Y lipolase lipolase L124 E129 Y164 lip2.1 Ra ID NO: 94) display HEYDMRVAW (SEQ Phage > E Y lipolase lipolase H215 E219 Y220 lip2.2 Ra ID NO: 95) display HEYPMDFHL (SEQ Phage > E Y lipolase lipolase H215 E219 Y220 lip2.2 Ra ID NO: 96) display SEYSMSITP (SEQ Phage > E Y lipolase lipolase S217 E219 Y220 lip2.3 Ra ID NO: 97) display CVWPAHAPLSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO: 98) display A P > S P208/P207 S214/S216/S217 CSWPSPAPLSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO: 99) display A P > S P208/P207 S214/S216/S217 CDFPLHAPLSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO: 100) display A P > S P208/P207 S214/S216/S217 CLFPSPAPRSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO: 101) display A P > S P208/P207 S214/S216/S217 CDGPAPAPWSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO: 102) display A P > S P208/P207 S214/S216/S217 CSFPLPAPRSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (SEQ ID NO: 103) display A P > S P208/P207 S214/S216/S217 CVYPSPAPWSCG Phage > P > > P lipolase lipolase P253 P250 A243 lip3.0 Ra (EQ ID NO: 104) display A P > S P208/P207 S214/S216/S217 PEYTMNALS (SEQ Phage > E Y lipolase lipolase P218 E219 Y220 lip2.4 Ra ID NO: 105) display CSRSAKGARLCG Phage > R S A lipolase lipolase R209 S214 A182 lip4.0-lac1.0- Ra (EQ ID NO: 106) display pd5.0- sav3.1-3.2 LEYPMSASQ (SEQ Phage > E Y lipolase lipolase L124 E129 Y164 lip2.1 Ra ID NO: 107) display RKLTLSGRS (SEQ Phage L > G R S lipolase lipolase L67 G65 R81 S83/S85 lip5.1-je4.0- Ra ID NO: 108) display sav9.0 RKLTLSGRS (SEQ Phage L > G R S lipolase lipolase L96/L97 G212 R209/ lip5.2-je4.0- Ra ID NO: 109) display R179 S214 sav9.0 SYGAPATPAA (SEQ Protein Poa p IX lipolase S170 Y171 G172 A173 lip6.0 Hu ID NO: 110) fragments P174 A150 T153 PAAGYTPAAP (SEQ Protein Poa p IX lipolase A18/A19/A20 G65 Y53 lip7.0 Hu Hu ID NO: 111) fragments T123 YKLAY (SEQ ID NO: Protein Poa p IX lipolase Y138 K74 L75 A68 Y16 lip8.1 Hu 112) fragments YKLAY (SEQ ID NO: Protein Poa p IX lipolase Y53 K127 L67 A68 Y16 lip8.2 Hu 112) fragments KYDDYVATLS (SEQ Protein Poa p IX lipolase Y194 D167 D165 Y164 lip9.0 Hu ID NO: 113) fragments V132 A131 L52 S54 EVKATPAGEL (SEQ Protein Poa p IX lipolase E43 V44 K46 A47 T72 lip10.0 Hu ID NO: 114) fragments CGYSNAQGVDYWI Protein Der p I lipolase Y53 S54 N25/N26 lip15.0 Hu Hu (SEQ ID NO: 115) fragments A18/A19/A20 Q15 V44 VPGIDPNACHYMKC Protein Der p II lipolase P256 I255 D254 P253 lip16.0 Hu (SEQ ID NO: 116) fragments N200 H198 Y261 SPVTKRASLKIDSKK Protein Der p II lipolase R179 A182 S216/S217 lip17.0 Hu (SEQ ID NO: 117) fragments I238 K237 I235 D234 S224 K223 IMSALAMVYLGAK Protein Ovalbumin lipolase V140 Y138 L69 A49 A47 lip18.0 Hu (SEQ ID NO: 118) fragments K46 ELGVRE (SEQ ID Protein a-amylase lipolase E99 L97 G109/G177 lip11.0 Hu NO: 119) fragments inhibitor V176 R175 D242 GCRKEV (SEQ ID Protein a-amylase lipolase G106 C107 R108 K98 lip12.0 Hu NO: 120) fragments inhibitor E99 LRSVYQ (SEQ ID Protein a-amylase lipolase L147 R81 S79 V77 Y16 lip13.0 Hu NO: 121) fragments inhibitor Q15 SGPWSW (SEQ ID Protein a-amylase lipolase S170 G172 P174 W89 lip14.0 Hu NO: 122) fragments inhibitor S83

TABLE 5 Amylase (Natalase) antibody binding peptide sequences, epitope patterns and epitope sequences. Antibody binding Method of peptide identification Epitope pattern Donor Acceptor Epitope Sequence ARIDPRGPS (SEQ Phage A > I D P R/K amylase amylase A380 K381 I382 D383 P384 ID NO: 123) display R389 ARIDPRHGS (SEQ Phage A > I D P R/K amylase amylase A380 K381 1382 D383 P384 ID NO: 124) display R389 CSVAKIDPRTCG Phage A > I D P R/K amylase amylase A109 K138 D140 P142 (SEQ ID NO: 125) display R144 CSVAKIDPRTCG Phage A > 1 D P R/K amylase amylase A380 K381 I382 D383 P384 (SEQ ID NO: 125) display R389 AKIDPKPDT (SEQ Phage A > I D P R/K amylase amylase A109 K138 D140 P142 ID NO: 126) display R144 AKIDPKPDT (SEQ Phage A > I D P R/K amylase amylase A380 K381 I382 D383 P384 ID NO: 126) display R389 ARIDPRHGS (SEQ Phage A > I D P R/K amylase amylase A109 K138 D140 P142 ID NO: 127) display R144 QIYNDTGPT (SEQ Phage Q > Y > D > amylase amylase Q390 L386 Y368/Y367 D366 ID NO: 128) display QIYNDTGPT (SEQ Phage Q > Y > D > amylase amylase Q170 I173 Y196 D195 ID NO: 128) display QIYNDTGPT (SEQ Phage Q > Y > D > amylase amylase Q357 I352 Y349 D366 ID NO: 128) display QIYNDTGPT (SEQ Phage Q > Y > D > amylase amylase Q331 I370 Y368/Y367 D366 ID NO: 128) display CGSATIDPRQCG Phage A > I D P R/K amylase amylase A109 K138 D140 P142 (SEQ ID NO: 129) display R144 CNADNQMYPQCG Phage A > > > > Y P > amylase amylase N29 A27 D26/D25 Y8 (SEQ ID NO: 130) display P41/P42 ARIDPRGPS (SEQ Phage A > I D P R/K amylase amylase A109 K138 D140 P142 ID NO: 131) display R144 CGSATIDPRQCG Phage A > I D P R/K amylase amylase A380 K381 I382 D383 P384 (SEQ ID NO: 132) display R389 CDADSSGYPLCG Phage A > > > > Y P > amylase amylase A107/A109 D108 Y57 (SEQ ID NO: 133) display P41/42 QLYGDEQLP (SEQ Phage Q > Y > D > amylase amylase Q331 I370 Y368/Y367 D366 ID NO: 134) display QLYGDEQLP (SEQ Phage Q > Y > D > amylase amylase Q357 I352 Y349 D366 ID NO: 134) display QLYGDEQLP (SEQ Phage Q > Y > D > amylase amylase Q170 I173 Y196 D195 ID NO: 134) display QLYGDEQLP (SEQ Phage Q > Y > D > amylase amylase Q390 L386 Y368/Y367 D366 ID NO: 134) display RYAQIDPRW (SEQ Phage A > I D P R/K amylase amylase A380 K381 I382 D383 P384 ID NO: 135) display R389 RYAQIDPRW (SEQ Phage A > I D P R/K amylase amylase A109 K138 D140 P142 ID NO: 135) display R144 GEFNLGRSS (SEQ Phage L > G R S amylase amylase L88 G92 R31 S28 ID NO: 136) display CNADSWGYPRCG Phage A > > > > Y P > amylase amylase N29 A27 D26/D25 Y8 (SEQ ID NO: 137) display P41/P42 CNADNQMYPQCG Phage A > > > > Y P > amylase amylase N102 A233 D232 Y54 (SEQ ID NO: 138) display P41/P42 CNADSWGYPRCG Phage A > > > > Y P > amylase amylase N102 A233 D232 Y54 (SEQ ID NO: 137) display P41/P42 GEFNLGRSS (SEQ Phage L > G R S amylase amylase L62 G63/G76 R78 S79 ID NO: 139) display Antibody binding peptide Epitope # IgG IgE ARIDPRGPS (SEQ je1.1 Ra ID NO: 123) ARIDPRHGS (SEQ je1.1 Ra ID NO: 124) CSVAKIDPRTCG je1.2 Ra (SEQ ID NO: 125) CSVAKIDPRTCG je1.1 Ra (SEQ ID NO: 125) AKIDPKPDT (SEQ je1.2 Ra ID NO: 126) AKIDPKPDT (SEQ je1.1 Ra ID NO: 126) ARIDPRHGS (SEQ je1.2 Ra ID NO: 127) QIYNDTGPT (SEQ je2.4 Ra ID NO: 128) QIYNDTGPT (SEQ je2.3 Ra ID NO: 128) QIYNDTGPT (SEQ je2.2 Ra ID NO: 128) QIYNDTGPT (SEQ je2.1 Ra ID NO: 128) CGSATIDPRQCG je1.2 Ra (SEQ ID NO: 129) CNADNQMYPQCG je3.1 Ra (SEQ ID NO: 130) ARIDPRGPS (SEQ je1.2 Ra ID NO: 131) CGSATIDPRQCG je1.1 Ra (SEQ ID NO: 132) CDADSSGYPLCG e3.3 Ra (SEQ ID NO: 133) QLYGDEQLP (SEQ je2.1 Ra ID NO: 134) QLYGDEQLP (SEQ je2.2 Ra ID NO: 134) QLYGDEQLP (SEQ je2.3 Ra ID NO: 134) QLYGDEQLP (SEQ je2.4 Ra ID NO: 134) RYAQIDPRW (SEQ je1.1 Ra ID NO: 135) RYAQIDPRW (SEQ je1.2 Ra ID NO: 135) GEFNLGRSS (SEQ je4.1-sav9.0-lip5.1-5.2 Ra ID NO: 136) CNADSWGYPRCG je3.1 Ra (SEQ ID NO: 137) CNADNQMYPQCG je3.2 Ra (SEQ ID NO: 138) CNADSWGYPRCG je3.2 Ra (SEQ ID NO: 137) GEFNLGRSS (SEQ je4.2-sav9.0-lip5.1-5.2 Ra ID NO: 139)

TABLE 6 Cellulase (Carezyme; Cel45 from Humicola insolens) antibody binding peptide sequences, epitope patterns and epitope sequences. Antibody binding Method of peptide identification Epitope pattern Donor Acceptor Epitope Sequence Epitope # IgG IgE CVHAGPRAGTCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 140) CVHAGPRAGTCG Phage display V H > G > carezyme carezyme car2.0 Ra (SEQ ID NO: 140) CLSGPLAGRVCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 141) CRISPWYSVPCG Phage display carezyme carezyme car3.0 Ra (SEQ ID NO: 142) CLSGPAAGQSCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 143) Phage display A > I D P R/K je-1 carezyme R146 I131 D133 car11.2 Ra P137 CITRGTRAGWCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 144) Phage display A R > A savinase carezyme A191 R200 R201 car6.2 Ra A83 N81 CLSGPAAGQSCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 143) A100 G101 Phage display A > I D P R/K je-1 carezyme A195 R37 I38 D40 car11.1 Ra L44 Phage display Q > Y > D > savinase, carezyme Q59 Y54 G134 D133 car10.0 Ra T136 je-1 Phage display > P > > A P > S lipoprime carezyme W62/W169 P61 P165 car9.0 Ra A162 P160 CITRGTRAGWCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 144) A100 G101 Phage display R/K R F > N savinase carezyme R7 R170 F174 car7.0 Ra A177 CLSGPLAGRVCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 145) A100 G101 Phage display A R > A savinase carezyme A1 R4 R7 A177 car6.1 Ra N176 Phage display > P > R D T G laccase carezyme D178 P180 R4 D2 car5.0 Ra S183 Phage display > R Y > K/R pd498 carezyme R170 R153 Y168 P165 car4.0 Ra K164 L163 Phage display D/E Q I F F T savinase carezyme Q36 I38 F41 F29 car8.0 Ra T197 CLTAGPSAGYCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 146) A100 G101 CYTTGRLAGLCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 147) CYTTGRLAGLCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 147) A100 G101 CVHSGPRAGYCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 148) CVHSGPRAGYCG Phage display V H > G >  carezyme carezyme car2.0 Ra (SEQ ID NO: 148) CVHAGPRAGTCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 149) A100 G101 CVHSGPRAGYCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 148) A100 G101 CVHSGLSRRLLR Phage display V H > G >  carezyme carezyme car2.0 Ra (SEQ ID NO: 150) CVTRGPNAGSCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 151) A100 G101 CLTAGPSAGYCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 152) CVTRGPNAGSCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 151) CITSGPRAGNCG Phage display > G > > A G carezyme carezyme T95/S96 G27 P98 car1.2 Ra (SEQ ID NO: 153) A100 G101 CITSGPRAGNCG Phage display > G > > A G carezyme carezyme P23 R201 A83 G84 car1.1 Ra (SEQ ID NO: 153)

TABLE 7 Laccase (Myceliophthora thermopila laccase) antibody binding peptide sequences, epitope patterns and epitope sequences. Antibody binding Method of Epitope peptide identification pattern Donor Acceptor Epitope Sequence Epitope # IgG IgE PQSD5PGESQ (SEQ Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra ID NO: 154) P165 G265 WPKSDAGDS (SEQ ID Phage display P > > D A G laccase laccase P241 R409 S410/S416 D434 lac4.1 Ra NO: 155) A389 G390 PQSDAGVVM (SEQ ID Phage display P > > D A G laccase laccase P241 R409 S410/S416 D434 lac4.1 Ra NO: 156) A389 G390 DPVRDTGAG (SEQ ID Phage display > P > R D T G laccase laccase P241 R409 D434 T432 lac5.1 Ra NO: 157) G430/G390 GPSRDAGLL (SEQ ID Phage display P > > D A G laccase laccase P241 R409 S410/S416 D434 lac4.1 Ra NO: 158) A389 G390 PASDAGRGP (SEQ ID Phage display P > > D A G laccase laccase P241 R409 S410/S416 D434 lac4.1 Ra NO: 159) A389 G390 PRDSTGLAL (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 160) P445 G446 PQSDPGESQ (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 161) P445 G446 RYPFLRATN (SEQ ID Phage display > R Y > K/R laccase laccase lac2.0- Ra NO: 162) pd1.1-1.4 GAARDARSA (SEQ ID Phage display > R S A laccase laccase lac1.0- Ra NO: 163) lip4.0- pd5.0- sav3.1- 3.2 PRSDTGFGS (SEQ ID Phage display > P > R D T G laccase laccase P241 R409 D434 T432 lac5.1 Ra NO: 164) G430/G390 LPRSDPGGR (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 165) P165 G265 DPARDTGDV (SEQ ID Phage display > P > R D T G laccase laccase P241 R409 D434 T432 lac5.1 Ra NO: 166) G430/G390 APKSDNGIT (SEQ ID Phage display P > > D A G laccase laccase P241 R409 S410/S416 lac4.1 Ra NO: 167) D434 A389 G390 PKSDPGTNW (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 168) P445 G446 PRTDPGWLA (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 169) P445 G446 LPRSDPGGR (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 170) P445 G446 PSSDPGARS (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 171) P165 G265 HVFDKNVTR (SEQ ID Phage display laccase laccase lac6.0 NO: 172) PRSDPGTPT (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 173) P445 G446 PRSDPGTPT (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 173) P165 G265 PRDSTGLAL (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 174) P165 G265 PRTDPGWLA (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 175) P165 G265 PSSDPGARS (SEQ ID Phage display P > S/T D P G laccase laccase P378 R379 T442 D443 lac3.1 Ra NO: 176) P445 G446 PKSDPGTNW (SEQ ID Phage display P > S/T D P G laccase laccase P180 R175 T168 D166 lac3.2 Ra NO: 177) P165 G265 WPKSDAGDS (SEQ ID Phage display P > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 178) PQSDAGVVM (SEQ ID Phage display P > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 179) GPSRDAGLL (SEQ ID Phage display P > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 180) PASDAGRGP (SEQ ID Phage display P > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 181) APKSDNGIT (SEQ ID Phage display P > > D A G laccase laccase P350 S349 D80 A79 G78 lac4.2 Ra NO: 182) WPKSDAGDS (SEQ ID Phage display P > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 183) PQSDAGVVM (SEQ ID Phage display P > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 184) GPSRDAGLL (SEQ ID Phage display P > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 185) PASDAGRGP (SEQ ID Phage display P > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 186) APKSDNGIT (SEQ ID Phage display P > > D A G laccase laccase P300 R234 S211 D213 A296 lac4.3 Ra NO: 187) DPVRDTGAG (SEQ ID Phage display > P > R D T G laccase laccase P378 R379 D469 T473 G446 lac5.2 Ra NO: 188) PRSDTGFGS (SEQ ID Phage display > P > R D T G laccase laccase P378 R379 D469 T473 G446 lac5.2 Ra NO: 189) DPARDTGDV (SEQ ID Phage display > P > R D T G laccase laccase P378 R379 D469 T473 G446 lac5.2 Ra NO: 190) DPVRDTGAG (SEQ ID Phage display > P > R D T G laccase laccase P60 R59 D51/D53 lac5.3 Ra NO: 191) T10/T12 G30 PRSDTGFGS (SEQ ID Phage display > P > R D T G laccase laccase P60 R59 D51/D53 lac5.3 Ra NO: 192) T10/T12 G30 DPARDTGDV (SEQ ID Phage display > P > R D T G laccase laccase P60 R59 D51/D53 lac5.3 Ra NO: 193) T10/T12 G30 DPVRDTGAG (SEQ ID Phage display > P > R D T G laccase laccase P157/P155 R23 lac5.4 Ra NO: 194) D118 T114 G113 PRSDTGFGS (SEQ ID Phage display > P > R D T G laccase laccase P157/P155 R23 lac5.4 Ra NO: 195) D118 T114 G113 DPARDTGDV (SEQ ID Phage display > P > R D T G laccase laccase P157/P155 R23 lac5.4 Ra NO: 196) D118 T114 G113

Example 2 Localisation of Epitope Sequences and Epitope Areas on the 3D-Structure of Acceptor Proteins

Epitope sequences were assessed manually on the screen on the 3D-structure of the protein of interest, using appropriate software (e.g. SwissProt Pdb Viewer, WebLite Viewer).

In a first step, the identified epitope patterns were fitted with the 3D-structure of the enzymes. A sequence of at least 3 amino acids, defining a specific epitope pattern, was localised on the 3D-structure of the acceptor protein. Conservative mutations (e.g. aspartate for glutamate, lysine for arginine, serine for threonine) were considered as one for those patterns for which phage display had evidenced such exchanges to occur. Among the possible sequences provided by the protein structure, only those were retained where the sequence matched a primary sequence, or where it matched a structural sequence of amino acids, where each amino acid was situated within a distance of 5 Å from the next one. Occasionally, the mobility of the amino acid side chains, as provided by the software programme, had to be taken in to consideration for this criterium to be fulfilled.

Secondly, the remaining anchor amino acids as well as the variable amino acids, i.e. amino acids that were not defining a pattern but were present in the individual sequences identified by phage library screening, were assessed in the area around the various amino acid sequences localised in step 1. Only amino acids situated within a distance of 5 Å from the next one were included.

Finally, an accessibility criterium was introduced. The criterium was that at least half of the anchor amino acids had a surface that was >30% accessible. Typically, 0-2 epitopes were retained for each epitope pattern. In some cases, two different amino acids could with equal probability be part of the epitope (e.g. two leucines located close to each other in the protein 3D-structure). For example, in Savinase two epitopes actually fit to the antibody binding peptide LDQIFFTRW (SEQ ID NO:62): L75 D41 Q2 179 and L42 D41 Q2 I79. A shorthand notation for such a situation is: L42/L75 D41 Q2 I79.

Thus, a number of epitope sequences were identified and localised on the surface of various proteins. As suggested by sequence alignment of the antibody binding peptides, structural analysis confirmed most of the epitopes to be enzyme specific, with only few exceptions. Overall, most of the identified epitopes were at least partially structural. However, some proteins (e.g. amylase) expressed predominantly primary sequence epitopes. Typically, the epitopes were localised in very discrete areas of the enzymes, and different epitope sequences often shared some amino acids (hot-spots).

The identified epitope sequences are shown in Tables 2-7.

Birch Allergen:

Bet v1 (WO 99/47680) was used as the parent protein for identification of epitope sequences that may cross react with enzyme epitopes. The structural coordinates from 1BV1.pdb (Gajhede et al., NAT.STRUCT.BIOL., Vol. 3, p. 1040, 1996) were used as well the corresponding sequence (Swissprot accession number P15494). The epitope pattern P>PAP>S (which had been identified from antibody binding peptides specific for anti-Lipolase antibodies) was found to match three (overlapping) epitope sequences on the surface of Bet v1:

Bet v1 1.1: P31 A34 P35 A37 P59 S39/S40; Bet v1 1.2: P63 L62 P59 A37 P35 S39/S40; and Bet v1 1.3: P59 S39/S40 P31 A34 P35 S39/S40. Example 3 Epitope Areas

It is common knowledge that amino acids that surround binding sequences can affect binding of a ligand without participating actively in the binding process. Based on this knowledge, areas covered by amino acids with potential steric effects on the epitope-antibody interaction, were defined around the identified epitopes. Practically, all amino acids situated within 5 Å from the amino acids defining the epitope were included. The accessibility criterium was not included for defining epitope areas, as hidden amino acids can have an effect on the surrounding structures.

For Savinase, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 2:

sav1.1 A1 Q2 S3 P5 H39 P40 D41 L42 N43 G63 T66 H67 A69 G70 T71 A73 A74 L75 N77 S78 I79 G80 V81 L82 G83 N204 V205 Q206 S207 T208 Y209 P210 S212 T213 Y214 A215 S216 L217 sav1.2 S153 G154 N155 S156 G157 A158 G160 S161 I162 S163 A169 R170 A174 M175 A176 V177 G178 R186 F189 S190 Q191 Y192 G193 A194 G195 L196 D197 I198 V199 T220 R247 K251 A254 T255 S256 T260 N261 L262 Y263 G264 S265 G266 L267 sav2.1 W6 G7 I8 R10 V11 Q12 A13 P14 A15 A16 R19 L21 V84 T180 D181 Q182 N183 N184 I198 V199 A200 P201 H226 V227 A230 L233 V234 K237 N238 H249 L250 T253 A254 T255 S256 L257 S265 G266 L267 V268 N269 A270 E271 A272 A273 T274 R275 sav2.2 S153 G154 N155 S156 G157 A158 S161 I162 S163 G178 A179 T180 D181 N184 N185 R186 A187 S188 F189 S190 Q191 Y192 G193 L196 T220 L262 Y263 sav2.3 A142 T143 G146 V147 L148 Y171 A172 N173 A174 M175 D197 A231 V234 K235 N238 P239 S240 W241 S242 N243 V244 Q245 I246 R247 N248 H249 L250 K251 sav3.1 S153 G154 N155 S156 G157 A158 V177 G178 A179 T180 D181 N184 N185 R186 A187 S188 F189 S190 Q191 Y192 V199 A200 P201 G202 V203 N218 G219 T220 A223 L262 Y263 sav3.2 L111 E112 G115 N116 M119 A138 V139 N140 S141 A142 S144 R145 G146 V147 V149 N173 N243 sav4.0 Q2 H17 T22 G23 S24 G25 V26 K27 V28 V30 I35 S37 T38 H39 P40 D41 L42 N43 I44 R45 G46 T66 A69 G70 T71 I72 A73 A74 L75 N76 N77 I79 G80 V81 L82 G83 V84 A85 P86 S87 A88 E89 L90 Y91 A92 T208 Y209 P210 S212 T213 Y214 sav5.1 A1 Q2 S3 V4 I35 S37 H39 P40 D41 L42 N43 I44 T66 A69 G70 A73 A74 L75 N76 N77 S78 I79 G80 V81 L82 G83 P86 L90 T208 Y214 sav5.2 V30 T33 G34 I35 S37 T38 L42 N43 I44 R45 G46 E54 S57 T58 Q59 D60 G61 N62 G63 H64 G65 T66 H67 A69 L90 Y91 A92 K94 P210 sav5.3 V4 P5 W6 G7 I8 S9 R10 V11 Q12 A13 P14 A15 A16 R19 N269 A270 E271 A272 A273 T274 R275 sav5.4 A1 Q2 P40 D41 F50 L75 N77 S78 I79 G80 V81 V104 S105 S106 I107 A108 Q109 G110 L111 E112 W113 A114 G115 N116 Q137 A138 S141 A142 Y214 sav6.1 V139 N140 T143 L148 V149 A151 P168 A169 Y171 A172 N173 A174 M175 A176 D197 I198 N243 V244 Q245 I246 R247 N248 H249 L250 K251 N252 T253 A254 S265 sav6.2 Q2 G25 V26 K27 V28 A29 I35 S37 T38 H39 P40 D41 L42 N43 I44 R45 G46 G47 Q59 T66 A69 G70 A73 A74 L75 N77 I79 G80 V81 L82 A88 E89 L90 Y91 N117 G118 M119 H120 V121 S207 T208 Y209 P210 G211 S212 T213 Y214 A215 sav7.1 K27 L31 I107 A108 Q109 G110 L111 E112 W113 A114 G115 N116 N117 G118 M119 A122 L124 L135 Q137 A138 V139 S141 A142 R145 V149 sav7.2 V104 I107 A108 L111 S132 A133 T134 L135 E136 Q137 A138 V139 N140 S141 A142 T143 S144 R145 G146 V147 V149 Y167 P168 Y171 A172 N173 A174 M175 N243 R247 sav9.1 L111 E112 A114 G115 N116 M119 H120 V121 A122 E136 Q137 A138 V139 N140 S141 A142 T143 S144 R145 G146 V147 L148 V149 V150 N173 M175 N243 I246 R247 L250 sav9.2 L126 G127 S128 P129 A152 S153 G154 S161 I162 S163 Y167 P168 A169 R170 Y171 A172 A176 V177 G178 Q191 Y192 G193 A194 G195 L196 D197 I198 V199 T260 N261 L262 Y263 G264 sav10.1 Q12 A13 P14 A15 A16 H17 N18 R19 G20 L21 T22 N76 L82 G83 V84 A85 P86 L233 V234 K237 N238 H249 L250 T253 N269 A270 E271 A272 A273 T274 R275 sav10.2 V11 Q12 A13 P14 A15 A16 H17 N18 R19 G20 L21 T22 G23 L233 V234 Q236 K237 N238 H249 L250 T253 A254 T255 L267 V268 N269 A270 E271 A272 A273 T274 R275 sav10.3 L31 D32 H64 V68 V95 L96 I107 L111 A114 G115 N116 M119 V121 A122 N123 L124 S125 L126 G127 S128 P129 V139 S141 A142 T143 S144 R145 G146 V147 L148 V149 V150 A151 A152 S153 S163 Y167 P168 A169 N173 A174 M175 A176 V177 T220 S221 M222 T224 P225 V227 A228 A231 N243 I246 R247 L250 sav10.4 P131 S132 A133 L135 E136 V139 A151 A152 S153 G160 S161 I162 S163 Y167 P168 A169 R170 Y171 A172 N173 A174 A176 Q191 Y192 G193 A194 G195 L196 R247 S259 T260 N261 L262 Y263 G264 sav11.0 W6 G154 N155 S156 G157 A179 T180 D181 Q182 N183 N184 N185 R186 A187 S188 F189 S190 Q191 Y192 P201 G202 V203 N204 V205 L217 N218 G219 T220 L262 Y263 sav12.0 L31 I107 A108 Q109 G110 L111 E112 W113 A114 G115 N116 N117 G118 A122 L124 S132 A133 T134 L135 Q137 A138 V139 N140 S141 T143 R145 V149 A151 S163 Y167 P168 A169 R170 Y171 N173 A174 sav13.0 Q2 S3 P5 T38 H39 P40 D41 L42 N43 H67 G70 A73 A74 L75 N77 I79 G80 V81 L82 G83 V205 Q206 S207 T208 Y209 S212 T213 Y214 A215 S216 L217 sav14.0 A16 H17 R19 G20 L21 T22 G23 S24 G25 V26 K27 V28 A29 V30 I35 I44 R45 G46 G47 V84 A85 P86 S87 A88 E89 L90 Y91 A92 V93 W113 N117 G118 M119 H120 V121 A232 L233 K235 Q236 K237 T274 sav15.0 W6 R10 G154 N155 S156 G157 V177 G178 A179 T180 D181 Q182 N183 N184 N185 R186 A187 S188 F189 S190 Q191 V199 A200 P201 G202 V203 N218 G219 T220 A223 L257 Y263 L267 sav16.0 A13 A16 H17 G20 L21 T22 G23 S24 G25 V26 V28 I72 A73 V84 A85 P86 S87 A88 E89 L90 H120 G229 A230 A231 A232 L233 V234 K235 Q236 K237 N238 P239 S240 W241 I246 H249 L250 A270 A273 T274 sav17.0 T22 G23 S24 G25 V26 K27 V28 A29 V30 L31 D32 I35 I44 R45 G46 G47 A48 F50 S87 A88 E89 L90 Y91 A92 V93 K94 V95 G110 W113 N117 G118 M119 H120 V121 A232 K235 Q236 sav18.1 W6 G7 I8 S9 R10 V11 Q12 A179 T180 D181 Q182 N183 N184 N185 R186 A187 I198 V199 A200 P201 V203 H226 V227 A230 H249 L250 K251 N252 T253 A254 T255 S256 L257 S265 G266 L267 V268 N269 A270 sav18.2 A13 A16 H17 L21 T22 G23 V26 V28 V84 A85 A88 V121 L148 Y171 A172 N173 V174 M175 A176 G195 L196 D197 I198 V199 V227 A228 G229 A230 A231 A232 L233 V234 K235 Q236 K237 N238 W241 N243 V244 Q245 I246 R247 N248 H249 L250 K251 N252 T253 A254 Y263 G264 S265 G266 V268 A270 A273 T274 sav19.1 A16 H17 R19 G20 L21 T22 G23 S24 G25 V26 K27 V28 S87 A88 E89 H120 V121 A232 L233 V234 K235 Q236 K237 N238 P239 T274 sav19.2 A1 Q2 S3 V4 P5 D41 H64 H67 G70 T71 A74 L75 N77 S78 I79 G80 V81 L82 G83 G202 V203 N204 V205 Q206 S207 T208 Y209 Y214 A215 S216 L217 N218 G219 M222

For PD498, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 3:

pd1.1 D105 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 N131 S132 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 W143 N144 K145 G146 A147 pd1.2 C128 E129 A153 G154 N155 D156 N157 V158 S160 R161 T162 F163 Q167 S170 G178 A179 I180 D181 D184 R185 K186 A187 S188 F189 S190 N191 Y192 G193 T194 W195 V196 T220 T262 N263 pd1.3 F50 L104 D105 S106 I107 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 W143 N144 K145 G146 A147 pd1.4 T28 *28aV A29 V30 D32 S33 G34 V35 Y37 *44aaV I45 K46 G47 Y48 D49 F50 I51 R53 D54 N55 N56 P57 M58 D60 L61 K89 I90 L91 A92 V93 R94 V95 L96 D97 A98 Y113 A114 Q117 A119 pd1.5 D32 S33 G34 K46 G47 Y48 D49 F50 I51 D52 R53 D54 N55 P57 M58 L61 L91 A92 V93 R94 V95 L96 D97 A98 L104 D105 S106 I107 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 G118 A119 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 pd2.1 V19 T21 I111 R112 Y113 A114 A1 D116 Q117 G118 A119 L122 D140 Y141 A142 W143 N144 K145 G146 A147 V148 L233 L234 A235 S236 Q237 G238 K239 N240 N243 V244 Q245 I246 R247 Q248 A249 A273 V274 R275 Y276 pd2.2 S24 S25 T26 Q27 T28 *28aV L42 A43 R44 *44aK *44aaV I45 D75 N77 D87 T88 K89 I90 L91 G118 A119 K120 V121 L122 G146 A147 V148 A232 A235 S236 pd2.3 R22 G23 S24 S25 T26 Q27 T28 *28aV D87 T88 K89 I111 A115 G118 A119 K120 V121 L122 S137 A138 V139 D140 Y141 A142 W143 N144 K145 G146 A147 V148 V149 V150 I175 A231 A232 A235 S236 N243 I246 R247 pd2.4 W-6 S12 T13 P14 A15 A16 V19 T21 R22 G23 S24 Q27 L230 A231 L233 L234 A235 S236 Q237 G238 K239 N240 N243 Q245 I246 S270 N271 K272 A273 V274 R275 Y276 pd3.1 L31 K46 G47 Y48 F50 L91 V93 S103 L104 D105 S106 I107 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 G118 L122 L124 C130 S132 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 Q167 P168 Y171 P172 pd3.2 V19 T21 R22 G23 S24 Q27 K120 V121 V148 L230 A231 A232 L233 L234 A235 S236 Q237 G238 K239 N240 N243 Q245 I246 R247 Q248 A249 I250 Q252 T253 K272 A273 V274 R275 Y276 pd4.1 W-6 S12 T13 P14 A15 A16 W17 D18 V19 T21 R22 G23 S24 M84 A85 P86 D87 T88 A142 W143 G146 A147 V148 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G238 K239 N240 N243 V244 Q245 I246 R247 Q248 A249 I250 S270 N271 A273 V274 R275 Y276 pd4.2 W-6 T13 A16 W17 V19 T21 R22 G23 S24 *44aK A73 A74 *75aT G83 M84 A85 P86 D87 T88 A142 G146 G146 A147 V148 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G232 K239 N240 N243 V244 Q245 I246 R247 Q248 A249 I250 S270 A273 V274 R275 Y276 pd4.3 T26 Q27 T28 *28aV A29 V30 L31 Y37 *44aaV I45 K46 G47 Y48 D49 D52 R53 D54 N55 N56 P57 M58 V72 T88 K89 I90 L91 A92 V93 Y113 A114 A115 Q117 G118 A119 K120 V121 L122 N123 A147 A228 A232 pd4.4 K46 G47 F50 L91 V93 S103 L104 D105 S106 I107 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 G118 C130 S132 T133 T134 L135 K136 S137 A138 V139 D140 Y141 Q167 P168 A169 S170 Y171 P172 N173 A174 pd4.5 T28 *28aV A29 V30 L31 V35 D36 Y37 N38 H39 L42 A43 *44aaV I45 K46 G47 Y48 F50 N55 N56 P57 M58 K89 I90 L91 A92 V93 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 G118 A119 L122 pd5.0 F50 S103 L104 D105 S106 I107 A108 S109 G110 I111 R112 Y113 A114 A115 D116 Q117 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 pd6.1 Y4 Y6 G7 G63 H64 H67 V68 T71 N155 A179 F189 P201 G202 V203 N204 I205 A206 S207 V209 G213 Y214 S215 Y216 M217 S218 G219 T220 S221 M222 A223 S224 P225 H226 pd6.2 W-6 T13 A16 W17 V19 T21 R22 G23 S24 S25 Q27 M84 A85 P86 D87 T88 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G238 S270 V274 pd7.0 R22 G23 S24 S25 Q27 T28 *28aV A29 V30 V35 D36 Y37 N38 H39 P40 D41 L42 A43 R44 *44aK *44aaV T66 A69 G70 V72 A73 A74 D75 N77 A85 P86 D87 T88 K89 I90 L91 A119 V121 L122 N123 T208 A228 A231 pd8.0 W-6 T13 A16 W17 T21 R22 G23 Q27 *44aK A73 A74 *75aT G83 M84 A85 P86 D87 T88 K120 V121 I175 A176 V177 G178 V196 D197 V198 T199 A200 V227 G229 L230 A231 A232 L233 L234 A235 S236 Q237 G238 K239 N240 N243 Q245 I246 Q248 A249 I250 Q252 T253 A254 F264 Y265 G266 I268 pd9.0 W-6 Y6 G7 P8 Q9 N10 T11 S12 T13 P14 A15 A16 W17 D18 V19 T21 M84 V139 W143 V148 V149 A151 P168 A169 Y171 P172 N173 A174 I175 A176 D181 S182 N183 D184 D197 P201 L230 L233 L234 K239 N240 N243 V244 Q245 I246 R247 Q248 A249 I250 E251 Q252 T253 A254 K267 I268 N269 S270 N271 K272 A273 V274 R275 Y276 pd10.0 L124 L126 G127 C128 E129 C130 N131 L135 V139 A151 A152 A153 G154 N155 D156 N157 V158 S160 R161 T162 F163 Q167 P168 A169 S170 Y171 A174 I175 A176 N191 Y192 G193 T194 W195 V196 T262 N263 F264 *264aK pd11.0 W-6 S-5 Y2 Y4 Q5 Y6 G7 P8 Q9 N10 T11 S12 T13 P14 W17 D18 V19 T21 A82 M84 I180 D181 S182 N183 D184 P201 G202 V203 N204 I205 H226 L233 S270 N271 V274 R275 pd12.0 G127 C128 E129 V139 V148 V149 V150 A151 A152 A153 G154 N155 D156 V158 R161 T162 F163 Q167 P168 A169 S170 Y171 P172 N173 A174 I175 A176 V177 G178 N191 Y192 G193 T194 W195 V196 D197 V198 T199 A200 V227 R247 I250 E251 A254 N263 F264 *264aK Y265 G266 I268 pd13.1 W-6 S-5 P-4 D-2 P-1 Y1 Y2 S3 *3aA Y4 Q5 P8 Q9 S12 T13 P14 A15 A16 W17 D18 V19 T21 R22 G80 V81 A82 N271 V274 R275 pd13.2 W-6 S-5 P-4 N-3 D-2 P-1 Y1 Y2 S3 *3aA Y4 Q5 P8 Q9 P14 W17 D41 G70 A74 D75 *75aT N76 N77 G78 I79 G80 V81 A82 G83 A206 S207 T208 Y214 pd14.0 T28 V35 D36 Y37 N38 H39 P40 D41 L42 A43 R44 *44aK *44aaV I45 K46 G47 Y48 D49 F50 R53 D54 N55 N56 P57 M58 T66 A69 G70 A73 A74 D75 K89 I90 L91 A92 V93 R94 Y113 T208 pd15.0 V30 L31 D32 S33 G34 V35 D36 Y37 N38 H39 L42 A43 *44aaV K46 Y48 D49 F50 I51 N56 P57 M58 D60 L61 N62 G63 H64 G65 T66 A69 I90 A92 V93 R94 V95 L96 D97 A98 G100 S101 G102 S103 S106 I107 G110 S125 L126 V209 P210 N211 N212 pd16.0 W-6 S-5 P-4 N-3 Y2 G7 P8 Q9 N10 T11 S12 T13 P14 A15 A16 W17 D18 V19 T21 R22 *75aT N76 A82 G83 M84 A85 P86 L233 N269 S270 N271 pd17.1 T11 S12 A15 A16 D18 V19 T21 R22 G23 S24 Q27 L230 A232 L233 L234 A235 S236 Q237 G238 K239 N240 N243 Q245 I246 Q248 A249 Q252 T253 N269 S270 N271 K272 A273 V274 R275 Y276 pd17.2 A108 I111 R112 A115 D116 K120 L124 T133 T134 L135 K136 S137 A138 V139 D140 Y141 A142 W143 N144 K145 G146 A147 V148 V149 P168 Y171 N173 A174 N243 pd18.1 W-6 T13 A16 W17 V19 T21 R22 G23 S24 S25 *44aK M84 A85 P86 D87 T88 K89 G229 L230 A231 A232 L233 L234 A235 S236 Q237 K239 A249 I250 T253 N269 S270 N271 K272 A273 V274 R275 Y276 pd18.2 D-2 V30 V35 D36 Y37 N38 H39 P40 D41 L42 A43 R44 *44aK *44aaV I45 K46 G47 Y48 P57 T66 A69 G70 A73 A74 D75 *75aT N76 N77 I79 V81 A82 A85 P86 D87 T88 K89 I90 L91 A92 V93 R94 T208

For Lipolase, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 4:

lip2.1 Y53 F55 V63 L78 F80 W117 V120 A121 D122 T123 L124 R125 Q126 K127 V128 E129 D130 A131 V132 R133 V140 L159 R160 G161 N162 G163 Y164 D165 I166 G190 lip2.2 V2 L6 F10 A173 P174 R175 A182 L193 Y194 R195 I196 T197 P204 R205 Y213 S214 H215 S216 S217 P218 E219 Y220 W221 I222 I235 V236 K237 I238 E239 I241 D242 A243 G246 N247 N248 lip2.3 V2 L6 F10 A182 L185 T186 L193 Y194 R195 I196 T197 H215 S216 S217 P218 E219 Y220 W221 I222 I235 V236 K237 I238 E239 G240 I241 A243 G246 N247 N248 lip2.4 V2 L6 F10 L193 Y194 R195 I196 T197 S216 S217 P218 E219 Y220 W221 I222 I235 V236 K237 I238 E239 G240 A243 G246 N247 N248 lip3.0 L93 K94 F95 H110 A173 P174 R175 V176 G177 N178 R179 A182 L185 T186 L193 R195 N200 D201 I202 P204 R205 L206 P207 P208 R209 E210 F211 G212 Y213 S214 H215 S216 S217 P218 E219 I238 E239 G240 I241 D242 A243 T244 G245 N248 ?R259? P250 N251 I252 P253 D254 I255 lip4.0 R175 V176 G177 N178 R179 A180 F181 A182 E183 F184 L185 T186 R205 P207 P208 R209 E210 F211 G212 Y213 S214 H215 S216 S217 I241 D242 N248 lip5.1 A20 Y21 N25 N26 T50 F51 L52 Y53 S54 F55 E56 V63 T64 G65 F66 L67 A68 L69 I76 V77 L78 S79 F80 R81 G82 S83 R84 S85 I86 E87 N88 W89 K127 V128 A131 H145 S146 L147 G148 L151 G266 lip5.2 K94 F95 L96 L97 K98 E99 R108 G109 H110 D111 G112 R175 V176 G177 N178 R179 A180 F181 A182 E183 F184 R205 P207 P208 R209 E210 F211 G212 Y213 S214 H215 S216 I241 D242 N248 lip6.0 Q9 F10 N11 F13 A14 S17 V63 F80 R81 W89 L93 F113 S116 W117 F142 T143 G144 H145 S146 L147 G148 G149 A150 L151 A152 T153 V154 A155 G156 A157 V168 F169 S170 Y171 G172 A173 P174 R175 V176 F181 L185 L193 Y194 R195 I196 T197 D201 V203 P204 L206 P207 H215 H258 Y261 F262 I265 lip7.0 F13 A14 Q15 Y16 S17 A180 A19 A20 Y21 C22 G23 N25 N26 I34 C36 A40 C41 F51 L52 Y53 S54 F55 E56 V63 T64 G65 F66 L67 S79 F80 R81 V120 A121 D122 T123 L124 R125 Q126 K127 V128 L264 I265 lip8.1 L12 F13 A14 Q15 Y16 S17 A18 A19 A20 I34 V44 A49 T50 F51 L52 F66 L67 A68 L69 D70 N71 T72 N73 K74 L75 I76 V77 S79 H135 P136 D137 Y138 R139 V140 V141 T143 lip8.2 L12 F13 A14 Q15 Y16 S17 A18 A19 A20 I34 V44 A49 T50 F51 L52 Y53 S54 F55 G65 F66 L67 A68 L69 D70 N73 L75 I76 V77 L78 S79 T123 L124 R125 Q126 K127 V128 E129 D130 A131 T143 lip9.0 L6 F10 N25 N26 D27 A28 A30 G31 T50 F51 L52 Y53 S54 F55 E56 G65 F66 L67 A68 L69 I76 T123 L124 R125 Q126 K127 V128 E129 D130 A131 V132 R1333 E134 H135 P136 R139 V140 V141 F142 G156 L159 R160 G161 N162 G163 Y164 D165 I166 D167 V168 F169 S170 G190 G191 T192 L193 Y194 R195 I196 Y220 lip10.0 N11 L12 Q15 Y16 I34 T35 C36 C41 P42 E43 V44 E45 K46 A47 D48 A49 D70 N71 T72 N73 K74 lip11.0 F95 L96 L97 K98 E99 I100 N101 D102 C107 R108 G109 H110 D111 F113 T114 S115 A150 T153 V154 A173 P174 R175 V176 G177 N178 R179 F181 V203 P204 R205 L206 P207 P208 R209 F211 G212 Y213 S214 H215 G240 I241 D242 A243 T244 N248 lip12.0 L96 L97 K98 E99 I100 N101 D102 C104 S105 G106 C107 R108 G109 H110 T114 S115 V176 G177 N178 A180 F181 F184 lip13.0 N11 L12 F13 A14 Q15 Y16 S17 A182 A19 A20 Y21 N26 I34 C36 A40 C41 P42 E43 V44 A49 F55 E56 V63 T64 G65 F66 L67 A68 D70 N73 L75 I76 V77 L78 S79 F80 R81 G82 S83 R84 W89 W117 L124 V128 V141 F142 T143 G144 H145 S146 L147 G148 G149 A150 L151 A152 A155 lip14.0 Q9 F10 N11 F13 A14 S17 Y21 R81 G82 S83 R84 S85 I86 E87 N88 W89 I90 G91 N92 L93 F113 T143 G144 H145 S146 L147 G149 A150 T153 V168 F169 S170 Y171 A173 P174 R175 V176 L193 Y194 R195 I196 T197 D201 V203 P204 L206 P207 H215 H258 Y261 F262 I265 G266 lip15.0 N11 L12 F13 A14 Q15 Y16 S17 A18 A19 A20 Y21 C22 G23 K24 N25 N26 D27 A28 I34 T35 C36 A40 C41 P42 E43 V44 E45 K46 A47 A49 F51 L52 Y53 S54 F55 E56 T64 G65 F66 L67 S79 F80 R81 T123 L124 K127 L264 I265 lip16.0 A14 E87 I90 H145 G172 I196 T197 H198 T199 N200 D201 I202 P204 R205 W221 I222 K223 S224 G225 T226 G246 N247 N254 I252 P253 D254 I255 P256 A257 H258 L259 W260 Y261 F262 G263 I265 lip17.0 E1 V2 F7 F10 G177 N178 R179 A180 F181 A182 E183 F184 L185 T186 L193 R195 H198 T199 G212 S214 H215 S216 S217 P218 E219 Y220 W221 I222 K223 S224 G225 T226 V228 P229 V230 T231 R232 N233 D234 I235 V236 K237 I238 E239 G240 I241 D242 A243 T244 G245 G246 I262 lip18.0 Q9 F13 Y16 T32 N33 I34 C41 P42 E43 V44 E45 K46 A47 D48 A49 T50 F51 L52 L67 A68 L69 D70 N71 T72 N73 L75 I76 V128 V132 H135 P136 D137 Y138 R139 V140 V141 F142 Y164 D165 I166 D167 F169 Y194

For Amylase, the following amino acid residues belong to the epitope area that correspond to each epitope sequence indicated in Table 5:

je1.1 N2 G3 T4 R33 P346 Y349 I352 L353 T354 R355 P360 V362 D366 Y367 M378 K379 A380 K381 I382 D383 P384 I385 L386 E387 A388 R389 Q390 N391 F392 A393 Y394 I450 T451 je1.2 Y57 D58 Y60 D61 F65 N66 Q67 L104 G105 G106 A107 D108 A109 T110 E111 A135 W136 T137 K138 F139 D140 F141 P142 G143 R144 G145 N146 T147 Y148 S149 F151 K152 W153 R154 F158 je2.1 M6 Y8 E10 W11 H12 D26 L30 R33 V325 D326 N327 H328 D329 S330 Q331 P332 G333 E334 E337 F339 K345 Y349 V362 F363 Y364 G365 D366 Y367 Y368 G369 I370 P371 T372 H373 S374 V375 P376 A377 M378 K379 I382 D383 L386 je2.2 L289 L293 V314 P318 T323 F324 V325 D326 F339 K345 P346 L347 A348 Y349 A350 L351 I352 L353 T354 R355 F356 Q357 G358 Y359 P360 S361 V362 F363 Y364 G365 D366 Y367 Y368 G369 P376 A377 M378 K379 I382 I385 R389 Q397 je2.3 N102 V116 E117 V118 P120 R123 D159 G160 V161 D162 W163 Q168 F169 Q170 N171 R172 I173 Y174 K175 A182 W183 D184 V187 D188 N193 Y194 D195 Y196 L197 M198 Y199 A200 D201 V202 H236 je2.4 T1 N2 T4 M6 Y8 D26 L30 R31 N32 R33 G34 I35 V325 D326 F339 K345 Y349 L353 V362 F363 Y364 G365 D366 Y367 Y368 G369 I370 P376 A377 M378 K379 I382 D383 P384 I385 L386 E387 A388 R389 Q390 N391 F392 Y394 H417 je3.1 M6 Q7 Y8 F9 E10 L13 H19 W20 N21 R22 L23 R24 D25 D26 A27 S28 N29 L30 R31 N32 R33 I385 W39 I40 P41 P42 A43 W44 V52 G53 Y54 Y75 A87 L88 N91 V93 D98 V100 Y364 Y368 je3.2 Y8 F9 W11 H19 W20 W39 I40 P41 P42 A43 W44 D51 V52 G53 Y54 G55 A56 Y75 D98 V99 V100 M101 N102 H103 L104 D195 L197 M198 A200 D201 V202 R230 I231 D232 A233 V234 K235 H236 I237 E262 H328 je3.3 Y8 F9 H19 W20 W39 I40 P41 P42 A43 W44 K45 G46 T47 V52 G53 Y54 G55 A56 Y57 D58 L59 Q67 K68 Y75 D98 V100 L104 G105 G106 A107 D108 A109 T110 E111 A135 W136 T137 K138 F139 D140 F141 P142 je4.1 L23 D25 D26 A27 S28 N29 L30 R31 N32 R33 G34 I35 T36 I38 A84 I85 H86 A87 L88 K89 N90 N91 G92 V93 Q94 V95 Q390 je4.2 A43 W44 K45 L59 Y60 D61 L62 G63 E64 F65 V71 R72 T73 K74 Y75 G76 T77 R78 S79 Q80 L81 E82 S83 Y148 W219 Y220 T223 L224

Example 4

Having identified ‘antibody binding peptide’ sequences and by consensus analysis also “epitope patterns” (e.g. >DF>>K>), one can identify potential epitope sequences on the 3-dimensional surface of a parent protein (=acceptor protein) in a semi-automated manner using the following method:

The anchor amino acid residues are transferred to a three dimensional structure of the protein of interest, by colouring D red, F white and K blue. Any surface area having all three residues within a distance of 18 Å, preferably 15 Å, more preferably 12 Å, is then claimed to be an epitope. The relevant distance can easily be measured using e.g. molecular graphics programs like Insight!! from Molecular Simulations Inc.

The residues in question should be surface exposed, meaning that the residue should be more than 20% surface exposed, preferably more than 50% surface exposed, more preferably 70% surface exposed. The percentage “surface accessible area” of an amino acid residue of the parent protein is defined as the Connolly surface (ACC value) measured using the DSSP program to the relevant protein part of the structure, divided by the residue total surface area and multiplied by 100. The DSSP program is disclosed in W. Kabsch and C. Sander, BIOPOLYMERS 22 (1983) pp. 2577-2637. The residue total surface areas of the 20 natural amino acids are tabulated in Thomas E. Creighton, PROTEINS; Structure and Molecular Principles, W.H. Freeman and Company, NY, ISBN: 0-7167-1566-X (1984).

Substitutions of one or more residue (s) within 18 Å, preferably 15 Å, more preferably 12 Å, around the geometrical center of the residues involved in the epitope, for a bigger or smaller residues, may destroy the epitope, and make the protein less antigenic.

Residues involved in epitope is 2, preferably 3 and more preferably 4

Example 5 Production, Selection, and Evaluation of Enzyme Variants with Reduced Antiqenicity or Immunogenicity

Epitope sequences and hot-spots amino acids were mutated using standard techniques know to the person skilled in the field (e.g. site-directed mutagenesis, error-prone PCR—see for example Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbour, N Y).

In the examples shown below, variants were made by site-directed mutagenesis. Amino acid exchanges giving new epitopes or duplicating existing epitopes, according to the information collected in the epitope-database (See Example 1), were avoided in the mutagenesis process.

Enzyme variants were screened for reduced binding of antibodies raised against the backbone enzyme. Antibody binding was assessed by competitive ELISA as described in the Methods section.

Variants with reduced antibody binding capacity were further evaluated in the mouse SC animal model (See methods section).

The following variants showed reduced IgE and/or reduced IgG levels in the mouse model:

Parent % IgG % IgE protein Mutations Target epitope sequences response response Savinase D181N Sav11.0; Sav15.0 and Sav18.1. 50 19 Hot spot amino acid. Savinase R170L; Q206E Sav9.4; Sav10.4; Sav1.1; and 5 34 Sav19.2 Savinase R170L, S57P Sav9.4; Sav10.4 45 12 Savinase R247E Sav2.3, Sav6.1, Sav18.2 75 30 Hot spot amino acid. Savinase R247Q Sav2.3, Sav6.1, Sav18.2 17 20 Hot spot amino acid. Savinase R247H Sav2.3, Sav6.1, Sav18.2 40 27 Hot spot amino acid. Savinase R247K Sav2.3, Sav6.1, Sav18.2 74 34 Hot spot amino acid.

Example 6 Production, Selection, and Evaluation of Enzyme Variants with Reduced Antigenicity or Immunogenicity

Hot-spots or epitopes were mutated using techniques known to the expert in the field (e.g. site-directed mutagenesis, error-prone PCR).

In the examples showed below, variants were made by site-directed mutagenesis. Amino acid exchanges giving new epitopes or duplicating existing epitopes according to the information collected in the epitope-database, were avoided in the mutagenesis process.

Enzyme variants were screened for reduced binding of antibodies raised against the backbone enzyme. This antibody binding was assessed by established assays (e.g. competitive ELISA, agglutination assay).

Variants with reduced antibody binding capacity were further evaluated in animal studies.

Mice were immunised subcutanuous weekly, for a period of 20 weeks, with 50 microliters 0.9% (wt/vol) NaCl (control group), or 50 microliters 0.9% (wt/vol) NaCl containing 10 micrograms of protein. Blood samples (100 microliters) were collected from the eye one week after every second immunization. Serum was obtained by blood clothing, and centrifugation.

Specific IgG1 and IgE levels were determined using the ELISA specific for mouse or rat IgG1 or IgE. Differences between data sets were analysed by using appropriate statistical methods.

A. Site-Directed Mutagenesis of Amino Acids Defining Epitopes, with an Effect on IgG1 and/or IgE Responses in Mice.

Epitope: A172/A169 R170A194 G193 N261 Pattern: AR>R>A>N Antibody: IgG1+IgE Backbone: Savinase

The variant carried the mutation R170F.

In a competitive IgE ELISA, this variant was less effective in competing for anti-savinase antibodies, giving a 15% lower endpoint inhibition as compared to the savinase backbone.

Mouse studies revealed an 80% reduction of the specific IgE levels, as compared to savinase backbone (p<0.01). The IgG1 levels were not significantly affected.

Epitope: S216 E219 Y220 Pattern: E Y>M Antibody: IgG1 Backbone: Lipoprime

The variant carried the mutation S216W.

In a competitive IgG ELISA, the variant was less effective in competing for Lipolase antibodies, giving a 38% decrease in endpoint inhibition as compared to the enzyme backbone.

Mouse studies revealed a 69% decrease in specific IgG1 levels, compared to the lipolase backbone (p<0.05). The IgE levels were not significantly affected.

B. Site-Directed Mutagenesis of Epitopes, with Examples of Epitope Duplication, and New Epitope Formation, Respectively, Predicted by the Epitope-Database.

Epitope: T143 N173 N140 E136 L135 Pattern: S/T NN>EL Antibody: IgG1 Backbone: Savinase

The variant carried the mutation E136R.

In a competitive IgG ELISA, the variants were less effective in competing for savinase antibodies, giving a 38% decrease in endpoint inhibition as compared to the savinase backbone.

Mouse studies revealed a dramatic increase in specific IgG1 levels, compared to savinase backbone (p<0.01). The IgE levels were not significantly affected.

Mutation E136R establishes an IgG1 epitope of the R Y P R/K pattern, previously identified on PD498. Apparently, this new epitope was more antigenic in mice than the existing epitope. The introduction of a savinase unrelated epitope on the savinase backbone could explain the observed discrepancy between competitive ELISA and animal studies.

In this example, it was found that using information derived exclusively from screening phage libraries with anti-PD498 antibodies (to identify the R Y P R/K epitope pattern of Table 2) one could predict the outcome of a genetic engineering experiment for Savinase in which the E136R mutation created the PD498-epitope on the Savinase surface, leading to increased immunogenicity of this Savinase variant. This demonstrates that the epitope patterns identified may be used to predict the effect on immunogenicity of substitutions in proteins that are different from the parent protein(s) used to identify the epitope pattern.

C. Site-Directed Mutagenesis of Amino Acids Defining Epitope Areas, with a Differential Effect on IgG1 and IgE Antibody Levels in Mice, and an Inhibiting Effect on IgG Binding, Respectively.

Epitope: A172/A169 R170A194 G193 N261 Pattern: AR>R>A>N Antibody: IgG1+IgE Backbone: Savinase

Epitope area: P131, S132, A133, L135, E136, V139, A151, A152, S153, G161, S162, I165, S166, Y167, P168, Y171, N173, A174, A176, Q191, Y192, G195, L196, R247, S259, T260, L262, Y263, G264.

The variant was different at position Y167 by the mutation Y167I.

In a competitive IgE ELISA, the variant was less effective in competing for anti-savinase antibodies, giving a 8% lower endpoint inhibition as compared to the its backbone.

Mouse studies revealed a 75% reduction of the specific IgE levels, as compared to the backbone (p<0.01). In contrast, the IgG1 levels were dramatically increased (p<0.01).

Epitope: T143 N173 N140 E136 L135 Pattern: S/T NN>EL Antibody: IgG1 Backbone: Savinase

Epitope area: V10A, 1107, A108, L111, E112, G115, S132, A133, T134, Q137, A138, V139, S141, A142, S144, R145, G146, V147, V149, Y167, P168, Y171, A172, A174, M175, N243, R247.

While variant no. 1 was mutated at the epitope position (N140D), variant no. 2 was mutated at N140 (N140D), but also at the epitope area position (A172D).

In a competitive IgG ELISA, variant no. 1 was less effective in competing for anti-savinase antibodies, as compared to savinase. This variant revealed a 21% lower endpoint inhibition as compared to the its backbone.

Variant no. 2 resulted in an endpoint inhibition that was 60% lower as compared to savinase, and 40% as compared to variant no. 1.

Example 7 Conjugation of Savinase Variant E136K with Activated Bis-PEG-1000

4.9 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 12 mg of N-succinimidyl carbonate activated bis-PEG 1000 in a reaction volume of approximately 2 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours.

The derivatives was purified and reagent excess removed by size exclusion chromatography on a Superdex-75 column (Pharmacia) equilibrated in 50 mM Sodium Borate, 5 mM Succinic Acid, 150 mM NaCl, 1 mM CaCl2 pH 6.0.

The conjugate was stored at −20° C., in the above described buffer.

Compared to the parent enzyme variant, the protease activity of the conjugate was retained (97% using Dimethyl-casein as substrate at pH 9).

Example 8

Competitive ELISA was performed according to established procedures. In short, a 96 well ELISA plate was coated with the parent protein. After proper blocking and washing, the coated antigen was incubated with rabbit anti-enzyme polyclonal antiserum in the presence of various amounts of modified protein (the competitior).

The amount of residual rabbit antiserum was detected by pig anti-rabbit immunoglobulin, horseraddish peroxidase labelled.

Epitope: T143 N173 N140 E136 L135 Pattern: S/T N N > E L Antibody: IgG1 Backbone: Savinase Mutation: E136K Modification: bis-NHS-PEG1000

The data show that the derivative (60% endpoint inhibition) has reduced capacity to bind enzyme specific immunoglobulines, as compared to the parent protein (100% endpoint inhibition).

Example 9

For this example the epitope sequences were determined in four environmental allergens (Bet v1; Der f2; Der p2 and PhI p2), based on their structures (1btv.pdb; 1ahm.pdb; a19v.pdb; and 1whp.pdb, respectively), sequences (SEQ ID NOS: 6, 7, 8 and 9, respectively) and computer modelling of the epitope patterns that had been assembled in our database (shown in Table 8). The allergens arise from common sources of allergy: Birch (Bet v1 from Betula pendula), House dust mites (Der f2 from Dermatophagoides farinae and Der p2 from Dermatophagoides pteronyssinus), and Timothy grass (PhI p2 from Phleum pratense).

The protein surface is scanned for epitope patterns matching the given “consensus” sequence of about 6-12 residues. First, residues on the protein surface that match the first residue of the consensus sequence are identified. Within a specified distance from each of these, residues on the protein surface that match the next residue of the consensus sequence are identified. This procedure is repeated for the remaining residues of the consensus sequence. The method is further described under the paragraph “Methods” above and the computer program can be found in the Appendixes.

The critical parameters used in this screening included:

    • i) a maximal distance between the alpha-carbon atoms of subsequent amino acids,
    • ii) a minimal accessibility of the amino acid of 20 Å2,
    • iii) the largest maximal distance between the most distinct amino acids should be less than 25 Å,
    • iv) the 5 best epitopes were taken,
    • v) the minimal homology with the epitope pattern of interest was 80%

In this way a number of potential epitopes are identified. The epitopes are sorted according to total surface accessible area, and certain entries removed:

    • 1) Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algorithm.
    • 2) Epitopes which are “too big”, i.e. where a distance between any two residues in the epitope exceeds a given threshold.

The epitope sequences found by this second generation mapping procedure were:

Bet v1: Epi#02 A146, K32, Q36, F30, T142, R145, V12 A34, K32, Q36, F30, T142, R145, V12 Epi#03 L62, K65, - - - , 156, Y66 L24, K20, H76, I23, Y81 L24, K20, H76, 1104, Y81 Epi#04 K134, S136, Q132, K129, A130, A135 K134, S136, Q132, K129, V128, G1 Epi#05 G140, A146, R145, T10, G111, A106, T107, V12 G26, A146, R145, T10, G110, A106, T107, V12 G140, A146, R145, T10, G110, S11, S149, L152 G110, A106, S11, T9, G140, R145, T10, V12 G140, A146, R145, T10, G111, S11, S149, V12 Epi#06 G110, P108, D109, T107, A106, P14 G111, P108, D109, T107, A106, P14 A34, N28, D27, S40, K32, P35 G26, N28, D27, S39, K32, P35 A106, N78, D75, T77, A16, P14 G26, N28, D27, S39, Q36, P35 Epi#07 G46, T52, D69, S99, R70, V71, P50, D72 G49, T52, D69, S99, R70, V71, P50, D72 G48, T52, D69, S99, R70, V71, P50, D72 Epi#08 K123, E127, G1, V2, H121, F3 K65, E60, F64, V67, F58 K65, E60, F58, V67, F64 K129, E127, G1, V2, H121, F3 Epi#09 S149, L152, D156, N159, R17, L24, D75, K103, N78, A106, V12 L152, S149, D156, N159, R17, L24, D75, H76, N78, A106, V12 L152, D156, N159, R17, L24, D75, K80, N78, A106, V12 Epi#10 D109, A106, N78, T77, F79, R17, K20 E141, T10, R145, T142, F30, G26, K32 E8, T10, R145, T142, F30, G26, K32 Epi#11 F30, K32, I38, Q36, V33, E148 F22, F30, I38, Q36, V33, E148 F30, L143, 138, Q36, V33, E148 Epi#12 Y5, E6 Y83, E73 Y120, E127 Y5, E8 Y66, E87 Y81, E73 Epi#13 H76, A16, P14, T107, A106, P108, G110, G111 A16, R17, P14, T107, A106, P108, G110, G111 A157, R17, P14, T107, A106, P108, G111, G110 Epi#15 K65, P90, D93, I91, K97, G92 K32, P31, D27, I56, K65, G61 Epi#17 A153, S149, R145, S11 A106, S11, R145, S149 Epi#18 R145, S149, L152, A153, Y150, L151, H154, S155 R145, S149, L152, A153, S155, L151, A157, N159 Epi#22 D125, D93, P90, K65 D93, P90, P63, E60 Epi#23 K55, N43, E42, S57, L62, P63 K68, N43, E42, S40, F30, P35 K54, N43, E42, S57, F64, P63 K55, N43, E42, S40, F58, P35 Epi#24 E96, K97, E87, P90, F64, E60, K65 E127, K123, E96, P90, F64, P63, K65 E42, K68, E87, P90, F64, E60, K65 E42, K55, E87, P90, F64, E60, K65 D93, G92, E87, P90, F64, E60, K65 D125, K123, E96, P90, F64, P63, K65 Epi#25 R70, K55, I44, E45, E42 R70, K54, I44, E45, N47 R70, K68, I53, E45, N47 Epi#27 D93, E127, D125, K123 Epi#28 A146, Q36, F58, E60, L62, F64, P63, K65 I38, Q36, F58, E60, L62, F64, P63, K65 A34, Q36, F58, E60, L62, F64, P63, K65 L143, Q36, F58, E60, L62, F64, P63, K65 V33, Q36, F58, E60, L62, F64, G61, K65 Epi#29 G61, K65, L62, F58, E60 I56, K65, L62, F64, E60 G89, K65, L62, F64, E60 V67, K65, L62, F64, E60 Epi#30 G1, N4, S99, H121, K97, I91, P90 I113, I13, S149, H154, S155, L152, L151 I13, L152, A153, H154, S155, L151, V33 G110, I13, S149, H154, S155, L152, L151 G1, N4, S99, H121, K97, I98, V2 G1, N4, S99, H121, K97, I91, V85 Epi#33 K32, F30, P35, S39, S57, K65 Q36, F30, P35, S39, S40, K32 K32, F30, P35, S40, S57, K65 K65, F58, P35, S39, A34, R145 Epi#34 V105, P14, T107, V12, R145, Y150, S155 I113, P14, T107, V12, R145, Y150, S155 Epi#37 P50, V74, L24, R17, N159 P50, V74, L24, K20, N159 P14, R17, L24, K20, N159 Epi#38 L143, G140, E141, R145, V33, N28, P31, S39 L143, G140, E141, R145, V33, N28, P31, S40 L143, G140, E141, R145, V33, N28, P31, S57 Epi#39 A130, E127, H126, T94, P90, G89, L62 A130, E127, H121, T94, P90, G89, L62 Epi#40 A157, L152, A153, Y150, K32, S39 A153, L152, A157, Y150, K32, S40 R17, L151, A153, Y150, K32, S40 R145, L143, A34, Y150, A153, S155 R145, L143, G140, T9, K115, T10 Epi#41 P63, Y66, L62, S57 Epi#44 I23, R17, D156, Y150, S149, V12, T10 L24, R17, D156, Y150, S149, V12, P14 L24, R17, D156, Y158, A16, A106, P108 I13, R17, D156, Y150, S149, V12, T10 L151, R17, D156, Y150, S149, V12, T10 L24, R17, D156, Y150, S149, V12, T107 Epi#45 K32, P35, F30, Y150, R145, M139, G140 K32, P35, F30, Y150, R145, M139, L143 K32, P31, F30, Y150, R145, M139, G140 Epi#47 L152, S149, R145, L143, A34, F30, N28, P31, P35 A153, S149, R145, A146, A34, F30, N28, P31, P35 Epi#48 E60, K65, P90, P63, G61 E60, K65, P63, P90, G92 Epi#51 T94, H126, E127, D125, G124, K123, H121 D125, H126, E127, T94, K123, T122, H121 Der f2: Epi#02 A98, K100, S101, P99, R128, R31 A98, K100, R128, P99, R31, V94 T91, N93, P95, P34, R31, R128 L61, N93, P95, P34, R31, R128 Epi#03 L40, K15, A39, I13, Y86 L40, K14, A39, I88, Y90 Epi#05 G32, A98, R31, P34, G20, T36, T91, Y90 G32, A98, R31, P34, G20, T36, T91, V94 G32, A98, R31, P34, G20, T36, T91, L37 G32, A98, R31, P34, G20, T36, T91, V18 Epi#06 A98, P99, D129, R31, K96, P95 G32, P99, D129, R128, R31, P95 A98, P99, D129, R31, K33, P95 A98, P99, D129, R31, K96, P34 A98, P99, D129, R128, K126, P26 Epi#07 T107, S57, D59, S101, R128, A98, P99, D129 T107, S57, D59, S101, R31, A98, P99, D129 Epi#08 K15, D87, V76, H74, F75 K14, D87, V76, H74, F75 K77, D87, V76, H74, F75 Epi#09 L61, D64, I68, H74, F75, T70, N71 N114, N46, D113, K48, N71, T70, T49 G83, N46, D113, K48, N71, T70, T49 Epi#10 L40, I13, D42, N44, V81, K48, N46, N114, G115 L40, I13, D42, N44, V81, K82, N46, N114, G115 L37, D19, G20, V18, V3, D4, K6, A120, T107, V105 Epi#11 F75, K51, I111, Q45, V116, D113 F75, K51, I111, Q45, V81, D113 Epi#12 Y90, E38 Epi#13 H30, R31, P95, A98, P99, S101, G60, L61 Epi#15

K96, P99, D129, 128, R128, A98

K96, P99, D129, I127, R128, A98 K96, P99, D129, I29, R128, A98 K55, P66, D64, I68, T70, G67 Epi#18 R31, R128, I28, G125, T123, H124, V105 R31, R128, I127, G125, T123, H124, V105 Epi#22 D1, M17, D4, V3, K6 D1, M17, D19, P34, K96 D1, M17, D4, V5, K6 Epi#23 K14, N11, E12, N44, Q85, P79 K14, N11, E12, N10, Q45, P79 K14, N11, E12, N44, Q84, P79 K14, N11, E12, L40, Q85, P79 Epi#24 D129, K100, E102, P99, R128, R31, K96 E62, G60, E102, P99, R128, R31, K96 D129, K126, E102, P99, R128, R31, K33 D129, K126, E102, P99, R31, P95, K96 Epi#25 R31, K96, I97, D59, E62 R128, R31, I97, D59, E102 R128, K126, I127, E102, N103 Epi#27 D64, E62, D59, K100 D59, E62, D64, K55 D87, E38, D19, K33 D19, E38, D87, K15 D19, E38, D87, K14 D19, E38, D87, K77 Epi#28 V16, D87, Q85, K14, E12, K15, Q2, D1 I13, D87, Q85, K14, E12, K15, Q2, D1 V3, D1, Q2, K15, E12, K14, Q85, D87 L40, D87, Q85, K14, E12, K15, Q2, D1 I88, D87, Q85, K14, E12, K15, Q2, D1 V76, D87, Q85, K14, E12, K15, Q2, D1 V18, D1, Q2, K15, E12, K14, Q85, D87 Epi#29 G32, N93, L61, E62 V94, N93, L61, E62 Epi#30 G60, I97, A98, H30, K96, P34, P95 I68, N71, H74, K77, P79, V81 G32, I97, A98, H30, K96, P95, P34 Epi#34 V105, P26, S24, G125, R128, S101, P99 W92, P34, T91, V94, R31, S101, P99 I28, P26, T123, G125, R128, S101, P99 Epi#37 A120, V16, L40, K14, N11 A39, V16, L40, K14, N11 Y90, A39, L40, K14, N11 Y86, A39, L40, K14, N11 Epi#39 A120, E38, T91, P34, G20, L37 A39, E38, T91, P34, G20, L37 Epi#40 G20, L37, A120, T123, K6, S24 A39, L37, A120, T123, K6, S24 G20, L37, A120, T107, K6, T123 Epi#41 P34, L37, V106, S57 Epi#42 P26, S24, G125, R128, R31 P99, S101, G125, R128, R31 Epi#44 V16, Q2, D19, P34, W92, Y90, A39, V18, T91 V16, Q2, D19, P34, W92, Y90, A39, V5, T123 V3, Q2, D19, P34, W92, Y90, A39, V18, T91 Epi#45 K77, H74, F75, N71, D69, G67

K77, H74, F75, N71, D69, V76

K77, H74, F75, N71, D69, V65 Epi#46 A98, R128, R31, P95, N93, G32 A98, R128, R31, P34, G20, Q2 Epi#48 Q2, D19, P34, P95, G32 H30, K96, P95, P34, G20 Epi#49 D87, D42, L40, Q85, Q84, C78, T47, Q45, K48 D87, D42, L40, Q85, Q84, C78, T47, Q45, K82 Epi#50 D19, W92, P34, T91 D19, W92, P34, P95 D19, W92, T91, T36 Epi#51 D129, H30, K33, R31, R128, K126, H124 R31, H30, D129, R128, K100, K126, H124 T123, H124, K126, R128, R31, K33, H30 Der p2: Epi#03 L17, K89, A39, I13, Y86 L17, K89, A72, I88, Y90 L17, K89, A72, I52, Y90 Epi#04 K15, S1, Q2, K14, V16, L17

K15, S1, Q2, K14, A39, L17

K15, S1, Q2, K14, V40, I13 Epi#05 G60, A56, L61, P99, G32, R31, H30, I97 G60, A56, L61, P99, G32, R31, H30, I28 Epi#06 G60, A56, D64, S57, K55, P66 G83, N46, D114, T49, K48, P79 G60, N103, D59, S101, R31, P95 Epi#08 K55, D64, S57, V106, F35 K55, E62, S57, V106, F35 Epi#09 L61, G60, E102, R128, I28, K126, N103, T123, V105 L61, G60, E102, R128, I127, K100, N103, T123, V105 L61, G60, E102, R128, I127, H124, N103, T123, V105 Epi#10 SAS: 435, Size 24.47: D69, T91, N93, F35, G32, R31 SAS: 422, Size 20.74: E38, T91, N93, F35, G32, K96 Epi#11 K14, I13, Q85, V81, E42 K15, I13, Q85, V81, E42 K14, I13, Q85, V40, D87 Epi#12 Y86, E42 Y90, E53 Y90, E38 Epi#13 H30, A125, P26, T123, A122, P19, L37, P34, W92 H30, A125, P26, T123, A122, H124, S24, G23, G20 H30, A125, P26, T123, A122, P19, L17, G20, F35 Epi#15 K55, P66, D69, I68, K89, A72 K55, P66, D69, I68, K89, A39 K55, P66, D64, I54, K109, G115 K55, P66, D64, I54, K109, A9 Epi#18 R31, I29, A125, S101, E102, N103 R31, I29, A125, S101, E102, V104 R31, I29, A125, T123, A122, V105 Epi#22 D69, P66, D64, V65, K55 D64, P66, D69, T91, K89 D59, L61, D64, P66, W92 D59, L61, D64, V65, E62 D69, P66, D64, V65, E53 Epi#24 D64, K55, E62, P99, R31, P34, K96 E53, K55, E62, P99, R31, P95, K96 D64, K55, E62, P99, R31, A98, K96 Epi#25 R31, H30, I28, E102, N103 R128, K126, I127, E102, N103 R128, K126, I28, E102, V105 Epi#27 D64, E53, D69, K89 D69, E53, D64, K55 D59, E62, D64, K55 Epi#28 V40, D87, Q85, E42, Q84, G83, K82 G20, H22, Q2, L17, E38, L37, Q36, P34, K33 G20, H22, Q2, L17, E38, L37, F35, P34, K33 Epi#29 I97, K100, L61, E62 G60, N103, L61, E62 I127, N103, L61, E62 Epi#30 G60, N103, S101, H30, K96, I97, P95 G60, N103, A125, H30, K96, I97, P95 I28, I127, A125, H30, K96, I97, P95 Epi#33 Q36, F35, V106, S57, A56, K55 K33, F35, V106, S57, A56, K55 Epi#34 I28, P26, S24, G23, G20, T123, S57 I28, P26, S24, V3, G20, T123, T107 W92, P34, T91, V18, G20, T123, P26 Epi#37 P66, V63, L61, K100, N103 P95, A98, L61, K100, N103 P19, V18, L17, K89, D87 P19, V3, L17, K89, D87 T123, V104, L61, K100, N103 Epi#38 L61, G60, E102, A125, V105, N103, P99, S57 L61, G60, E62, A56, V105, N103, P99, S57 Epi#39 A125, E102, H124, T123, P26, G20, L17 Epi#40 G60, L61, A56, T107, K6, T123 A39, L17, G20, T123, P26, S24 G60, L61, A56, T107, K55, S57 G60, L61, A56, T123, K126, S101 Epi#41 P19, L17, V3, S1 P19, L17, V5, S24 Epi#44 V65, D64, P66, W92, Y90, A39, V18, P19 L61, D64, P66, W92, Y90, A39, V18, T91 Epi#45 R31, P34, F35, N93, V94 K96, P34, F35, N93, G32 Epi#47 I127, S101, R31, I97, A98, L61, N103, P99, P95 I28, S101, R31, I97, A98, L61, N103, P99, S57 Epi#48 H30, K96, P95, P99, G60 H30, K96, P34, P19, G20 H30, K96, P34, P19, V18 H30, K96, P34, P95, V94 H30, K96, P34, P19, V3 E38, K89, P70, P66, V65 H30, K96, P95, P34, G32 Q36, K89, P70, P66, V65 Epi#50 D69, Y90, W92, P66, P70 D69, Y90, W92, P34, P95 D69, Y90, W92, T91, P34 D69, Y90, W92, V94, P95 D69, Y90, W92, L37, P19 Epi#51 K126, H124, E102, R128, I28, R31, H30 T123, H124, K126, R128, I28, R31, H30 D4, H124, K126, R128, I28, R31, H30 PhI p2: Epi#02 T87, K85, Q61, S38, R34, R67 T87, K85, Q61, P63, R34, V42 Epi#03 K10, A90, I88, Y86 K10, A18, I88, Y86 Epi#04 R34, S38, Q61, K85, T87, I88 R34, S38, Q61, K85, T87, A90 Epi#05 G47, A18, S12, T87, G89, T91, T5, V1 G73, A29, L69, T27, G50, T53, T45, V42 G11, A18, L20, T91, G89, A90, T87, I88 Epi#06 A93, P94, D79, R34, Q61, P59 A93, P94, D79, R34, Q61, P83 A93, P94, D80, R34, Q61, P59 A93, P94, D79, R34, Q61, P63 Epi#08 K10, E9, G11, A18, H16, F54 K46, E48, G47, A18, H16, F54 K10, E9, S12, A18, H16, F54 Epi#09 L69, T27, G73, N76, R67, V77, D79, R34, A43, T45, V42 L69, T27, A29, E30, R67, V77, D80, R34, A43, T45, V42 Epi#10 D55, A18, N13, S12, F54, G47, K46 T45, A18, N13, S56, F54, G47, K46 Epi#09 L60, S56, E57, D55, K15, N13, S12, G11 L60, S56, E57, D55, H16, F54, T45, T53 L60, S56, E57, D55, H16, F54, T45, G47 Epi#12 Y86, E84 Y23, E24 Epi#18 N76, R67, F78, V81, A93, Y92, T91, T5, P2, V1 Epi#19 D39, W41, S38, Q61, R34, G37 E40, W41, S38, Q61, R34, A43 Epi#22 D79, P94, D80, P83, K85 D79, P94, D80, P63, K85 Epi#23 K10, N13, E14, L60, Q61, P59 K10, N13, E14, L60, Q61, P83 K10, N13, E14, L60, Q61, P63 Epi#24 E58, K15, E57, P59, S56, E14, Q61 D55, K15, E57, P59, S56, E58, Q61 Epi#25 R34, R67, W41, D39, E40 Epi#26 S38, E40, W41, V42, E32, E30 S38, E40, W41, V42, A43, E32 Epi#27 E14, E57, E58, K15 D55, E14, E84, K85 Epi#28 G37, H36, Q61, K85, E84, L60, F54, A43, K46 G37, H36, Q61, K85, E84, L60, F54, S12, D55 G37, H36, Q61, K85, E84, L60, F54, S56, D55 G37, H36, Q61, K85, E84, L60, F54, A43, R67 G37, H36, Q61, K15, E57, L60, F54, A43, K46 G37, H36, Q61, K85, E84, L60, F54, S12, K15 G37, H36, Q61, K85, E84, L60, F54, S56, K15 G37, H36, Q61, K85, E84, L60, F54, A43, R34 G37, H36, Q61, K85, E84, L60, F54, A18, D55 Epi#29 G73, K72, L69, R67, E30 I88, N13, L60, F54, E57 G25, K72, L69, R67, E32 V77, K75, L69, R67, E30 G37, H36, L60, F54, E57 G37, Q61, L60, F54, E57 Epi#30 I88, N13, S12, H16, K15, P59, L60 I88, N13, S56, H16, K15, L60, P59 I88, N13, A18, H16, K15, P59, L60 Epi#33 K46, F54, V42, S56, K15 H16, F54, V42, S56, K15 Epi#34 V1, P2, T5, V4, P94, Y92, T87 V1, P2, T5, L20, G89, T91, T87 V81, P94, T5, V1, P2, Y92, T91 Epi#37 T27, A29, L69, K72, D26 A43, R67, L69, K75, N76 Epi#38 L20, G89, E9, A18, N13, P59, S56 Epi#40 G49, L20, G89, Y86, K85, T87 G49, L20, G89, T87, K10, S12 G49, L20, G89, T87, K10, T7 Epi#44 V77, R67, D79, P94, Y92, A93, V1, P2 L69, R67, D79, P94, Y92, A93, V1, T5 Epi#45 D79, P94, F78, N76, M74, L69 D80, P94, F78, R67, D79, V77 K3, P94, F78, N76, M74, G73 Epi#46 A43, R67, R34, P63, H36, Q61 V77, R67, R34, P63, H36, G37 L69, R67, R34, P63, G37, Q61 Epi#47 G37, E35, E40, A43, R34, L60, N13, P59, S56 V77, E32, E40, A43, R34, L60, N13, P59, S56 S38, G37, E40, A43, R34, L60, N13, P59, S56 Epi#48 E24, K3, P94, P2, V1 E84, D80, P94, P2, V1 Epi#50 D39, W41, A43, T45 D39, W41, V42, T45 Epi#51 D79, H36, E84, T87, K10, G11, H16 D39, H36, Q61, K85, P63, R34, W41 D79, H36, E40, D39, G37, R34, W41 Q61, H36, E84, T87, K10, G11, H16

TABLE 8 Each row indicates an epitope pattern. At each position (from 1 to maximum of 12) the cells indicate which amino acids (single letter coding) are allowed at that position. The last column indicates the patterns identified using IgE antibody binding. Epitope Pattern Position Number 1 2 3 4 5 6 7 8 9 10 11 12 1 TS RQ YS NHC KR KR P HNP L IgE 2 RV R Y- PST FR- ALPQS- RKN ALT IgE (SEQ ID NO: 197) 3 Y I AH- K L 4 AGIL ANRTV- KRY Q S Y- KR (SEQ ID (SEQ ID NO: 198) NO: 199) 5 GILVY STH ASTR- G PT- RNAFLS A G IgE (SEQ ID (SEQ ID (SEQ ID NO: 200) NO: 201) NO: 202) 6 P KRQSA STRC D PAN GA IgE (SEQ ID (SEQ ID NO: 203) NO: 204) 7 D P AV- R S- D S- T G 8 F HI- VA- FSG- DE- KA IgE 9 NRGLTV- STAN ANF RKH D- AILV- R- (ENRSV- AGI- DGNT- LIS- IgE (SEQ ID (SEQ ID (SEQ ID SEQ ID (SEQ ID NO: 205) NO: 206) NO: 207) NO: 208) NO: 209) 10 KR RG F C- AST- RN NTA DECT IgE (SEQ ID NO: 210) 11 DE V- Q I FLK F 12 E Y IgE 13 FWYGL PG ALS- PH A T- P LRWA SAH IgE (SEQ ID (SEQ ID NO: 211) NO: 212) 14 GV Q ILV I- Y GNR DN TEH 15 AG RKQT- I D P RKN IgE (SEQ ID NO: 213) 16 DN A DA SDN QRSW GMR Y P RQL (SEQ ID NO: 214) 17 S- R S A 18 VLSFN AEHNPT- T- L- ST- Y- GAL LIV- CSF- R FRN- SD IgE (SEQ ID (SEQ ID NO: 215) NO: 216) 19 AGLKM R Q QSC NTW DEI IgE (SEQ ID NO: 217) 20 D G D KN L LF- P K V A IgE 21 P S I- I LR- CI IgE 22 EDKW ACLPTV D- ASLPM- D- IgE (SEQ ID WY- (SEQ ID NO: 218) (SEQ ID NO: 220) NO: 219) 23 AP LQF SYLN- E N RK IgE (SEQ ID NO: 221) 24 KQ AELFPR- TSFR P EA GK DE IgE (SEQ ID (SEQ ID NO: 222) NO: 223) 25 ENV DE IW- RKH R IgE 26 DE AGE PHV W E- S W IgE 27 K DE E DE IgE 28 DKR APSG- QF- CFIKLW- E FIKLW- Q DH- AGILV IgE (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 224) NO: 225) NO: 226) NO: 227) 29 E RF- L KRQHNG GILV IgE P (SEQ (SEQ ID ID NO: NO: 229) 228) 30 LVP LIP IKLPQS- H AS- LIMN GI (SEQ ID (SEQ ID NO: 230) NO: 231) 31 D FI- MV- FW R N QR L 32 V f- DE A A F 33 KR SA- S VP YF KQH IgE 34 STP STY GPR- GLV STM WP IVW 35 I M S A- L AG 36 AW A PV- K- Q- ST Y- G- V- A A TP IgE 37 NYD KR L ARV TYAP IgE (SEQ ID NO: 232) 38 S P N LR- RV- AR- E G L 39 L G P RT- HL- E A 40 ST- APK- YT AG L- AGR IgE 41 St V- L Yh- P- 42 RQ R P- H- NQG S P L 43 T- RI ML S HQ GL YA WC I 44 PT AGV SA Y W- P- D- RQ- ILVS IgE (SEQ ID NO: 233) 45 LVG MD- RN Y- F PH KRD IgE 46 AGQ HNQGC P R R AVLCY IgE (SEQ ID (SEQ ID NO: 234) NO: 235) 47 PS RP N LFQA- AR AILMNV RE AGSYLE LIAGVS (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 236) NO: 237) NO: 238) NO: 239) 48 GV P P KHQD SHQE (SEQ ID (SEQ ID NO: 240) NO: 241) 49 KN Q- TMC WYC- Q Q FP- VP- L W- D 50 PST STAPLW W WY- RHD V (SEQ ID NO: 242) 51 WH TSKHRQ LIRKGP DSRTQG DEKQHT H RKQDT G (SEQ (SEQ ID KH- (SEQ (SEQ ID (SEQ ID ID NO: NO: 244) ID NO: NO: 246) NO: 247) 243) 245) 52 Q DNT- W R STRE- A FW (SEQ ID NO: 248)

Example 10

For this example the third-generation epitope sequences were determined in further 11 environmental allergens (Bosd2, Equc1, Gald4-mutant (with alanine substituted for glycine in position 102), Hevb8, Profillin1-AC, Profillin1-AT, Profillin2-AC, Profillin-birch pollen, Rag weed pollen5 and Vesv5), based on their structures sequences (SEQ ID NOS: 12, 13, 15, 16, 17, 18, 19, 20, 21 and 22, respectively), their structures (1bj7.pdb, 1ew3.pdb, 1flu.pdb, 1g5u.pdb, 1prq.pdb, 1a0k.pdb, 1f2k.pdb, 1cqa.pdb, 1bbg.pdb, and 1qnx.pdb, respectively), and computer modelling of the epitope patterns that had been assembled in our database (shown in Table 8). Further, the epitope sequences of the four environmental allergens of example 9, Bet v1, Der f2, Der p2, and PhI p2, were redetermined.

The additional allergens arise from common sources of allergy: cows (Bos d2 which is a bovine member of the lipocalin family of allergens), horses (Equ C1, a major horse allergen also of the lipocalin family), Hen egg white (Lysozyme Gal D 4), Latex (Hey b8, a profilin from Hevea brasiliensis), Acanthamoeba castellani (Profilin1-AC, a profilin isoform IA and Profilin2-AC, a profilin isoform II), Arabidosis thaliana (Profillin1-AT a cytoskeleton profilin), Birch (Profilin-birch pollen (Birch pollen profilin), Rag weed pollen5 (Ragweed pollen allergen V from Ambrosia trifida) and whasp venom (Ves v5 allergen from Vespula vulgaris venom).

The protein surface is scanned for epitope patterns matching the given “consensus” sequence of about 6-12 residues. First, residues on the protein surface that match the first residue of the consensus sequence are identified. Within a specified distance from each of these, residues on the protein surface that match the next residue of the consensus sequence are identified. This procedure is repeated for the remaining residues of the consensus sequence. The method is further described under the paragraph “Methods” above and the program can be found in Appendixes.

The critical parameters used in this screening included:

    • i) a maximal distance between the alpha-carbon atoms of subsequent amino acids,
    • ii) a minimal accessability of the amino acid of 20 Å2,
    • iii) the largest maximal distance between the most distinct amino acids should be less than 25 Å,
    • iv) the best epitope were taken,
    • v) the homology with the epitope pattern of interest was 100%

In this way a number of potential epitopes are identified. The epitopes are sorted according to total surface accessible area, and certain entries removed:

    • a. Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algorithm.
    • b. Epitopes which are “too big”, i.e. where a distance between any two residues in the epitope exceeds a given threshold.

The epitope sequences found were:

Bosd2: Epi#01 L65, P155, P156, R17, R40, N37, Y39, R41, T67 L65, P155, P156, R17, R40, N37, Y39, R41, S52 L64, P155, P156, R17, R40, N37, Y39, R41, T54 Epi#02 T121, K150, S122, R17, P156, Y39, R41, R40 T121, K150, S122, R17, P156, Y56, R36, V30 Epi#03 L128, K130, H92,17, Y76 L134, K130, H92,17, Y76 L128, K130, H92,191, Y76 Epi#04 R72, Y76, S50, Q73, K71, V69, I45 K71, Y76, S50, Q73, R72, V69, L80 K71, Y76, S50, Q73, R72, V69, I42 Epi#06 G14, P13, D47, S10, K11, P9 G14, P13, D47, S10, S94, P9 G14, P13, D47, C44, S10, P9 Epi#08 K71, E49, S50, V69, F82 K71, E49, S50, V79, F82 Epi#09 I7, S10, D8, E95, K119, N96, S122, T121 S10, I7, D8, E95, K11, N96, S122, T124 Epi#10 E15, T54, R41, T67, F55, R17, K119 E43, T54, R41, T67, F55, R17, K119 E31, T151, N153, C63, F55, R40, R41 E31, T151, N153, C154, F55, R41, R17 Epi#11 K26, I145, Q132, E143 K26, I145, Q132, E137 K26, I145, Q132, E129 Epi#12 Y105, E108 Y83, E81 Epi#15 N153, P156, D152, I149, T121, G120 R17, P156, D152, I149, T121, G120 N153, P156, D152, I149, R17, G14 Epi#18 R109, 1110, G107, Y83, T85, E81, V69 R109, 1110, G107, Y105, T85, E81, V69 Epi#19 E43, N46, S50, Q73, R72, K71 D47, N46, S50, Q73, R72, G75 E49, N46, S50, Q73, R72, K71 I45, N46, S50, Q73, R72, K71 Epi#20 V30, K28, P34, L57, L65, K58, D59, G32, D27 V30, K28, P34, L57, L64, K58, D59, G33, D27 Epi#22 D8, S10, D47, P13, E15 D8, S10, D47, P13, E43 D47, S10, D8, V93, E95 D8, S10, D47, C48, K71 Epi#23 K119, N96, E127, S122, L128, P125 K150, N147, E146, Y20, F123, P125 K11, N96, E127, S122, L128, P125 Epi#24 E129, K130, E126, P125, S122, L128, Q133 E126, K130, E129, P125, S122, R17, K119 E126, K130, E129, P125, T124, L128, Q133 Epi#25 R72, K71, I45, D47, N46 R72, K71, I45, E43, N46 Epi#27 D47, E49, E74, K71 D24, E143, E146, K150 D47, E43, E15, K119 Epi#28 L134, Q133, L128, E126, K130, F123, S122, K150 Q132, K130, E126, L128, F123, S122, K150 L65, D59, Q60, K58, E31, L57, G32, D27 G61, D59, Q60, K58, E31, K28, G32, D27 Epi#29 V69, K71, L80, R72, E74 I45, K71, L80, R72, E74 G61, Q60, L64, F55, E68 Epi#30 G120, N96, S94, H92, K130, L128, P125 I91, I7, S94, H92, K130, L128, P125 Epi#33 K130, F123, P125, S122, K150 K71, Y76, P9, S10, S94, K119 Epi#34 I7, P9, S10, G14, R17, T121, S122 I45, P13, S10, G14, R41, Y39, P156 Epi#37 T67, V69, L80, K71, Y76 P156, R40, L65, K58, D59 P155, R40, L65, K58, N153 Epi#38 L80, G84, E108, R109, N25, P141, S136 Epi#39 E137, R138, P141, G139, L134 E31, L57, R36, P34, G84, L80 Epi#40 R17, G120, T121, K150, S122 R17, G120, T121, K150, T151 Epi#41 P34, Y83, L80, V69, S52 P34, Y83, L80, V79, S50 Epi#42 L128, P125, S122, G120, R17, R41 L128, P125, S122, G120, R17, R40 Epi#44 S10, D47, P9, Y76, S50, V69, T67 I45, D47, P9, Y76, S50, V69, T67 Epi#45 D27, P34, F82, Y105, R109, D106, G107 D59, P34, F82, Y105, R109, D106, G107 K58, P34, F82, Y105, R109, D106, G107 D27, P34, F82, Y105, R109, D106, G84 Epi#46 Y39, R41, R40, P155, C63, Q60 Y20, R17, R40, P155, C63, Q60 Epi#47 L128, E126, E129, L134, R138, Q133, N142, P141, S136 V69, E81, E68, I42, R41, F55, N37, R40, P156 V69, E43, E15, I42, R41, F55, N37, R40, P156 S122, E127, E129, L134, R138, Q133, N142, P141, S136 Epi#48 E43, D47, P13, P9, V93 S10, D47, P9, P13, G14 E43, D47, P13, P9, V90 E49, D47, P13, P9, V93 Equc1: Epi#02 L66, N68, A65, F90, S69, Y72, R64, V89 A65, R64, S31, F28, S112, Y123, R110, V108 L179, R180, Q178, F177, P143, Y38, R141, V145 L66, R64, S31, F28, S112, Y123, R110, V125 L66, N68, A65, F90, S69, Y72, R64, V62 Epi#03 K32, A65, I63, Y72 Epi#05 G35, A65, S69, T93, G97, R26, S112, Y123 G35, A65, S69, T93, G97, R26, S112, I25 Epi#07 G97, T93, S70, D91, S100, R110, V125, P132, D128 Epi#08 K129, D130, F127, V108, F90 K129, D130, F127, V108, F109 K129, D130, F127, V125, F136 K129, D130, F127, V125, F133 Epi#10 E48, N53, N80, T77, C83, F177, R175, K172 E82, N80, N53, T77, C83, F177, G181, R180 E52, N53, N80, T77, C83, F177, R175, K172 Epi#11 F133, K47, I167, Q158, V163, E165 Epi#12 Y38, E142 Y38, E36 Y139, E142 Epi#13 K129, P132, D45, 1167, Q158, G161 R131, P132, D45, 1167, K164, G161 Epi#16 P87, Y72, R64, S70, S69, D67, A65, N68 Epi#17 A65, S31, R64, S34 Epi#18 R64, S31, I30, A65, S34, L66, N68, S69 Epi#19 E82, N80, C83, Q178, R175, K172 Epi#22 D130, P132, D128, Y106, K129 Epi#23 D144, K150, E148, P147, S146, E151, K155 Epi#25 R160, K159, I156, E151, E148 Epi#27 E118, E142, D144, K172 E36, E142, D144, K172 Epi#28 I173, D174, Q178, L179, E85, C83, F177, G181, R180 I173, D174, Q178, L179, E85, C83, F177, P143, D144 Epi#29 G181, Q178, L179, R180, E36 G181, Q178, L179, R180, E85 Epi#30 I30, N27, S112, H119, I121, I25, V23 Epi#31 L122, R110, N27, R26, F28, I30, D96 L124, R110, N27, R26, F28, I30, D96 Epi#33 H119, Y38, V62, S34, S31, R64 Epi#34 V62, P87, M88, V89, R64, S31, S34 Epi#37 P87, V89, L66, R64, D67 Epi#40 R64, L66, A65, Y72, S34 R64, L66, A65, Y72, S69 Epi#41 P132, Y106, L101, V89, S100 P132, Y106, L101, V89, S70 Epi#44 V46, R131, D128, P132, Y106, S100, V89, P87 Epi#45 K129, P132, F127, Y106, N102, D91, V89 K129, P132, F127, Y106, N102, D104, G105 Epi#47 S146, E148, E152, V23, R26, A24, N27, R110, S112 V23, E115, E118, N116, R26, F28, N27, R110, S112 Gald4: Epi#01 L75, N65, P70, R73, R61, N59, Y53, R45, T47 L75, N65, P70, R68, R61, N59, Y53, R45, T47 Epi#02 A90, N77, L75, R73, P70, R61, R68 A122, R125, Q121, T118, R114, R112 Epi#04 R21, Y20, S24, Q121, R125, R128, L129 R21, Y20, S24, Q121, R125, R128, G126 Epi#05 G16, A10, R128, G126, A122, T118, G117 G4, A10, R128, G126, A122, T118, G117 Epi#06 G67, P79, D66, R61, R73, P70 G67, N65, D66, S72, R73, P70 G49, N46, D48, R61, R73, P70 Epi#07 G71, T69, D66, S72, R73, P70, D48 G67, T69, D66, S72, R73, P70, D48 Epi#08 K1, D87, S86, V2, F38 K1, D87, S86, V2, F3 Epi#09 Epi#10 E7, A11, R14, A10, C6, F3, R5, R125 D87, A11, R14, A10, C6, F3, R5, R125 T47, N46, N44, S36, F34, R114, R112 D18, A10, R14, A11, C6, F3, R5, R125 T118, N113, R112, A110, F34, R114, K116 Epi#11 L129, I124, Q121, V120, D119 Epi#12 Y53, E35 Epi#15 R73, P70, D66, I78, A82 R73, P70, D66, I78, A90 Epi#17 A102, S100, R21, S24 Epi#18 R112, N113, R114, F34, V109, A107, A102, N103 N113, R112, R114, F34, V109, A107, N103, S100 Epi#19 D18, N19, S24, Q121, R125, L129 D18, N19, S24, Q121, R125, G126 Epi#22 D48, P70, D66, W63, W62 D66, P70, D48, T69, W62 D48, P70, D66, W63, K97 Epi#23 R45, N44, E35, N39, Q41, A42 R45, N44, E35, Y53, Q41, A42 Epi#25 R128, R125, W123, D119, N27 R128, R125, W123, D119, V120 Epi#26 W62, S72, W63, P79, A82, D87 W62, S72, W63, P79, G67, D66 Epi#28 G117, D119, Q121, I124, E7, C6, F3, A11, R14 A122, D119, Q121, I124, E7, C6, F3, A11, R14 Epi#29 G126, R125, L129, R128, E7 G16, R14, L129, R128, E7 Epi#30 I124, L129, A10, H15, I88, L84 I124, L129, A11, H15,188, L84 Epi#31 L75, R73, N65, R61, W62, I98, D101 L75, R73, N74, R61, W62, I98, D101 Epi#33 Q41, F38, V2, S86, S85, K1 Q41, F38, V2, S36, A110, R114 Epi#34 W63, W62, T69, G71, R73, S72, P70 W62, W63, S72, L75, R73, T69, P70 Epi#36 A110, A107, A102, S100, K96, A90, A82 Epi#37 A10, R128, L129, R14, D18 A10, R128, L129, K13, N19 Epi#40 R128, L129, A11, T89, A90, S85 R14, L129, A11, T89, A90, S85 Epi#41 Y53, L84, S81 Y53, L84, S86 Epi#42 P79, S81, N65, P70, R61, R73 P79, S81, N65, P70, R61, R68 Epi#44 L129, R14, D18, Y20, S24, V120, T118 L129, R14, D18, Y23, S24, V120, T118 Epi#46 L75, R61, R73, P70, N65, G67 L75, R73, R61, P70, N65, A82 L75, R61, R68, P70, N65, G67 Epi#47 S72, G71, R68, N65, R61, L75, N77, R73, P70 G67, S72, R68, N65, R61, L75, N77, R73, P70 Epi#49 D87, L84, Q41, Q57, Y53, T43, N44 D87, L84, Q57, Q41, Y53, T43, N46 D87, L84, Q41, Q57, Y53, T43, N39 Epi#50 R73, W62, W63, P79, S81 R73, W63, W62, S72, P70 Epi#51 D18, H15, K13, R14, L129, R125, W123 Epi#52 F34, A110, R112, R114, W111, N27, Q121 F3, A11, E7, R5, W123, D119, Q121 W123, A122, T118, R114, W111, N27, Q121 Hevb8: Epi#01 L20, P109, P112, K86, R84, N116, Y125, Q129, T111 L110, P109, P112, K86, R84, N116, Y125, Q129, T111 Epi#02 A48, K43, Q41, F42, T70, Y72, R84, V74 T21, R19, P109, P112, R84, V74 A49, K43, Q41, F42, T70, Y72, R84, V74 Epi#03 L65, K86, I75, Y72 Epi#05 G30, A48, L60, P62, G58, T63, H66, G69 G58, A61, R84, P112, G113, T111, S89, G88 G80, A81, F54, P79, G58, T63, H66, G69 G77, A81, F54, P79, G58, T63, H66, G69 Epi#06 G58, P79, D55, S59, K52, P57 G80, P79, D55, S59, K52, P57 G77, P79, D55, S59, K52, P57 Epi#07 G17, T5, S2, D16, R19, P109, D107 Epi#08 K52, D45, S44, A49, H66, F42 Epi#10 E78, A81, R96, F54, G58, K52 D55, A81, R96, F54, G80, K52 Epi#11 F54, L60, I83, Q76, V82, E78 Epi#12 Y106, E108 Epi#13 H66, L65, P62, T63, A61, P57, A81, P79, G58 H66, L65, P62, T63, A61, P57, A81, P79, G80 H66, L60, P62, T63, A61, P57, A81, P79, G77 Epi#15 R19, P109, D107, I105, K86, G88 Epi#18 R19, G17, P109, S89 Epi#22 D29, S44, D45, A48, K52 D29, M51, D55, P79, E56 D45, M51, D55, P79, E78 D29, S44, D45, A49, K52 D45, M51, D55, P79, E56 D29, M51, D55, P57, E78 D29, M51, D55, P57, E56 D45, M51, D55, P57, K52 D45, M51, D55, P57, E78 Epi#24 D55, K52, E56, P79, F54, E78, Q76 D45, K52, E56, P57, F54, E78, Q76 Epi#25 R84, K86, I105, D107, E108 R96, H28, I26, D29, V3 Epi#26 W33, S2, W3, V32, G30, D29 Epi#27 D53, E56, D55, K52 Epi#28 V32, Q41, K43, E46, K52, F54, P57, D55 G69, Q41, K43, E46, K52, F54, P57, D55 Epi#29 G130, Q99, L127, R96, E78 L127, Q99, L131, R96, E78 G98, Q99, L127, R96, E78 Epi#30 G69, L67, A49, H66, K71, L65, P62 G80, M51, A48, H28, Q99, L127, L131 Epi#33 Q41, F42, V32, S31, S44, K43 Q41, F42, V47, S44, A48, K52 Q41, F42, V47, S44, A49, K52 Epi#34 I105, P112, S89, L110, R19, T21, S37 I105, P112, T111, L20, R19, T21, S37 Epi#37 T63, A49, L60, K52, D55 P62, V74, L60, K52, D45 P62, A61, L60, K52, D55 Epi#38 G77, E78, R96, V82, R84, N116, P112, S89 Epi#39 A48, E46, H66, T63, P62, G58, L60 A49, E46, H66, T63, P62, G58, L60 Epi#40 R19, L110, G113, T111, P109, S89 R19, L110, G113, T111, P112, S89 Epi#41 P62, L65, V47, S44 P109, Y106, L110, S89 P112, Y106, L110, S8 Epi#44 L20, R19, D16, W3, Y6, S2, G17, P109 L110, R19, D16, W3, Y6, S2, G17, P109 Epi#45 K52, P57, F54, R96, D124, L127 D55, P79, F54, R96, D124, L131 Epi#47 I75, G77, E78, V82, R84, N116, P112, S89 I75, G77, E78, I83, R84, N116, P112, P109 Epi#48 E78, Q76, P79, P57, G58 E78, Q76, P79, P57, G80 E78, Q76, P79, P57, G77 Epi#50 D9, W3, W33, S2, T5 D16, W3, W33, S2, T5 Epi#51 R19, H18, E108, S89, K87, K71, H66 R19, H18, E108, D107, K87, K71, H66 Profillin1-AC: Epi#01 L116, N111, P106, K80, K81, N101, S83, Q105, T108 L116, N111, P106, K80, K81, N101, Y100, Q105, S83 Epi#02 T44, N51, P54, R56, T69, Y78, R71, V68 L24, K93, S92, R75, S76, Y78, R71, R56 Epi#03 L24, K93, I121, Y119 L24, K90, I121, Y119 Epi#04 K80, Y100, S83, Q105, K103, N101, G82 K80, Y100, S83, Q105, K103, T17, G12 K80, Y100, S83, Q105, K103, T17, G14 Epi#05 G34, A33, A36, T38, G64, A63, H66, V68 G34, A33, S32, T17, G12, T4, S1, Y5 Epi#06 A46, N50, D53, R56, A57, P54 A52, N50, D53, R56, A57, P54 A72, N50, D53, R56, A57, P54 A57, P54, D53, S47, Q43, P39 Epi#07 G64, T38, D61, S58, R56, A57, P54, D53 G64, T38, D61, S58, R56, A52, P54, D53 Epi#08 K103, E102, G82, V68, H66, F60 K81, E102, G82, V68, H66, F60 Epi#09 L24, S47, D53, A57, V68, R71, L70, R56, N51, N50, R75 L24, S47, D53, A57, V68, R71, L70, R56, N51, T44, T38 Epi#10 D74, N50, N51, T44, F60, R56, R71 D53, N50, N51, T44, F60, R56, R71 Epi#11 F125, K93, I121, Q123, D118 F125, K90, I121, Q123, D118 F49, K90, I121, Q123, D118 Epi#12 Y119, E114 Y100, E102 Epi#13 A57, R56, P54, T44, A40, P39, A36, G64, Y67 S58, A57, P54, T44, A40, P39, A36, G64, Y67 Epi#15 N51, P54, D53, I55, R56, A57 R56, P54, D53, I55, T69, A57 R56, P54, D53, I55, T44, A40 Epi#16 Q105, P106, Y100, G14, Q18, S32, A36, A33, D7 Q105, P106, Y100, G14, Q18, S32, A36, A63, D61 Epi#17 A110, S76, R75, S92 A72, S76, R75, S92 Epi#18 N51, N50, R75, S92, L24, S47, T44, P39, N27 N51, N50, R75, S92, L24, T28, T38, P39, N27 Epi#22 D53, S47, D25, L24, K93 D53, S58, D61, V68, K81 Epi#23 K103, N101, E102, S83, Q105, P106 K103, N101, E102, S83, Q105, A84 Epi#24 E114, K115, A110, P106, S83, E102, K103 D53, G59, A57, P54, R56, L70, K80 E102, K103, A15, P106, S83, A84, Q105 Epi#25 R71, R56, 155, D53, N50 R71, R56, 155, D53, N51 Epi#28 I104, Q105, K103, E102, K81, S83, K80 G107, Q105, K103, E102, K81, G82, K80 A84, Q105, K103, E102, K81, S83, K80 A110, Q105, K103, E102, K81, S83, K80 Epi#29 I121, K115, L116, E114 V112, K115, L116, E114 Epi#30 G59, I55, S58, H66, K80, L70, V68 G59, I55, S58, H66, K80, P106, V99 Epi#33 K80, Y78, V68, S58, A57, R56 K81, Y67, V68, S58, A57, R56 Epi#34 I55, P54, S58, V68, R71, Y78, P106 W29, W2, T4, V11, G12, Y5, S1 Epi#36 A63, A36, A33, V11, G14, Y100, S83, Q105, K103, P106, A110, A15 A63, A36, A33, V11, G14, Y100, T108, Q105, K103, P106, A15, A110 Epi#37 A57, R56, L70, R71, Y78 A57, V68, L70, R56, D53 Y78, R71, L70, R56, N51 P54, R56, L70, R71, D73 T69, R71, L70, R56, D53 Epi#38 G82, E102, A84, V99, N101, P106, S83 Epi#40 R71, L70, A72, Y78, K80, S83 R71, L70, G59, T69, K81, S83 R56, L70, A72, T69, K81, S83 Epi#41 P106, Y78, L70, V68, S58 Epi#42 P54, S47, N51, R56, R71 P54, S58, G59, R56, R71 Epi#44 S83, Q105, P106, Y78, A110, G107, T108 V68, R71, D73, Y78, A110, G107, T108 L70, R71, D73, Y78, A110, V112, T108 L70, R71, D73, Y78, A110, G107, P106 Epi#45 K81, H66, F60, R56, D53, G59 K80, H66, F60, R56, D53, G59 D61, H66, F60, R56, D53, G59 Epi#46 L70, R71, R56, P54, N51, A52 L70, R71, R56, P54, N51, A72 V68, R71, R56, P54, N51, A46 Y78, R71, R56, P54, G59, A57 Epi#47 V68, A57, R56, L70, R71, A52, N51, P54, S58 S58, A57, R56, L70, R71, A72, N51, P54, S47 Epi#49 D25, L24, Q43, Q41, T44, N51 D25, L24, Q43, Q41, T38, N27 Epi#50 D7, W2, W29, S1, T4 D7, Y5, W2, W29, S1 Epi#51 K80, H66, D61, T44, P39, T28, W29 K80, H66, D61, T38, P39, T28, W29 Profillin 1-AT: Epi#01 P109, P89, K86, R84, N116, Y106, Q114, T111 Epi#02 L42, K43, Q45, F66, T63, Y72, R84, V74 L42, K43, Q45, F66, T63, Y72, R84, V82 Epi#03 K96, I127, Y125 K86, I75, Y72 Epi#05 G77, A81, F54, P57, G58, A61, T63, V74 G58, A61, F59, P57, G77, A81, T97, G80 G80, A81, F54, P57, G58, A61, T63, Y72 Epi#06 G17, P109, D107, T21, K38, P40 G112, P109, D107, T21, K38, P40 G88, P89, D107, T21, K38, P40 Epi#08 K52, E55, G58, V74, F66 K51, E55, G58, A61, F59 Epi#09 D29, D48, K52, F59, A61, T63 D29, D48, K51, F59, A61, T63 Epi#10 E108, T111, N18, T21, F39, G68, K71 E108, T111, N18, T21, F105, G112, K86 Epi#11 F105, K86, I75, Q76, V82, E78 F66, K43, I47, Q28, V32, D29 F59, K52, I47, Q28, V32, D29 Epi#12 Y125, E130 Y125, E128 Epi#15 K43, P44, D29, I47, K52, G58 K43, P44, D48, I47, Q45, G49 K43, P44, D29, I47, K51, G80 Epi#20 K38, P40, F39, L42, K43, D48, G30, D29 K51, P57, F59, L60, K52, D48, G30, D29 Epi#22 D48, P44, D29, V32, W33 D48, P44, D29, V32, W3 Epi#24 D29, K51, E56, P57, F59, E55, Q79 D48, K52, E55, P57, F59, E56, Q79 Epi#25 R121, K95, I83, D53, E55 R121, K95, I83, E78, V82 Epi#26 W33, S2, W3, V32, G30, D29 Epi#27 E128, E130, D124, K96 E130, E128, D124, K95 Epi#28 I75, Q76, E78, Q79, P57, K51 A61, Q76, E78, Q79, P57, K52 V32, D29, Q99, E130, I127, S129, D124 V32, D29, Q99, 1127, E128, S129, D124 Epi#29 V32, Q41, L42, F66, E70 G69, Q41, L42, F66, E70 G68, Q41, L42, F66, E70 Epi#30 G17, N18, H19, Q114, L117, V15 G17, M110, H19, Q114, L117, V15 G113, M110, H19, Q114, L117, V15 Epi#33 Q41, F39, P40, S36, A37, K38 Epi#34 V74, P62, M73, G88, P89, Y106, T111 Epi#37 T111, V15, L117, R121, Y125 T111, V15, L117, R121, D124 Epi#39 A81, E55, P57, G58, L60 A81, E78, P57, G58, L60 Epi#40 R121, L117, G112, Y106, P109, T111 R121, L117, G112, Y106, P89, T111 Epi#41 Y125, L131, S129 Epi#44 I75, R84, Y72, A61, G58, P62 I75, R84, Y72, A61, V74, T63 Epi#45 K38, P40, F105, Y106, N18, D14, G17 K38, P40, F105, Y106, N18, D107, G88 K38, P40, F105, Y106, N18, D14, V15 Epi#48 E16, H19, P109, P89, G88 E16, H19, P109, P89, G112 Epi#49 D124, L131, Q99, Q28, T97, N98 D124, L131, Q99, Q28, T97, K96 Epi#50 D9, Y6, W3, W33, S2 D9, W3, W33, S2, S5 D9, W3, W33, V32, S31 Epi#51 D14, H19, E108, T111, L117, R121, H10 D107, H19, E16, Q114, L117, R121, H10 D14, H19, D107, T21, K38, Q35, W33 Profillin2-AC: Epi#01 L116, N111, P106, K80, K81, N101, S83, Q105, T108 L116, N111, P106, K80, K81, N101, S83, Q105, T108 Epi#02 T53, N58, S57, R56, T69, Y67, R66, V68 T53, K50, A52, R56, T69, Y67, R66, V68 T53, K50, A72, R56, T69, Y67, R66, V68 Epi#03 L116, K115, I121, Y119 Epi#04 K81, Y100, S83, Q105, K103, T17, G12 K80, Y100, S83, Q105, K103, A84, G82 K81, Y100, S83, Q105, K103, T17, G14 K80, Y100, S83, Q105, K103, N101, I104 K81, Y100, S83, Q105, K103, A15, G107 Epi#06 A54, N47, D25, T28, A36, P39 A40, N27, D25, T28, A36, P39 A44, N47, D25, T28, A36, P39 G34, A33, D7, T31, A36, P39 A43, N47, D25, T28, A36, P39 Epi#08 K103, E102, G82, V68, F60 K103, E102, G82, V68, F60 K81, E102, G82, V68, F60 Epi#10 T53, N58, R56, S57, F60, R66, K81 E61, N58, R56, S57, F60, R66, K80 Epi#11 F125, K93, I121, Q105, E102 F125, K93, I121, Q123, D118 Epi#12 Y100, E102 Y119, E114 Epi#13 A52, A44, P39, A43, H24, S92, G124, Y119 A46, A44, P39, A43, H24, S92, G124, Y119 Epi#15 K103, P106, D118, I121, K93, G124 K103, P106, D118, I121, Q105, G107 K103, P106, D118, I121, Q123, G122 Epi#16 Q105, P106, Y78, R71, S57, N58, A54, A44, D51 Q105, P106, Y78, R71, R56, D51, D74, A52, N47 Epi#18 R66, N58, R56, S57, V68, G82, S83, E102, N101 R66, N58, R56, S57, V68, G82, S83, P106, N101 Epi#22 D74, A52, D51, T53, K50 D25, A44, D51, T53, K50 D74, A46, D51, T53, K50 D74, A72, D51, T53, K50 Epi#23 K103, N101, E102, S83, Q105, P106 K103, N101, E102, S83, Q105, A84 Epi#24 D74, K81, A84, P106, S83, E102, K103 D74, K81, E102, P106, T108, A15, K103 Epi#25 R66, K81, E102, N101 Epi#28 I121, D118, Q105, K103, E102, K81, G82, D74 G107, D118, Q105, K103, E102, K81, G82, D74 G122, D118, Q105, K103, E102, K81, G82, D74 Epi#29 I121, K115, L116, E114 V112, K115, L116, E114 Epi#30 I55, N47, A44, H24, K93, I121, L116 I55, N47, A43, H24, K93, I121, L116 Epi#31 R56, N58, R66, F60, V68, I55, D51 R66, N58, R56, F60, V68, I55, D51 Epi#33 K115, Y119, P106, S83, A84, K103 Q123, Y119, P106, S83, A84, K103 K81, Y67, V68, S57, A54, R56 K80, Y78, V68, S57, A54, R56 Epi#34 W29, W2, T8, V11, G12, T4, S1 W29, W2, T4, G12, G14, T13, T8 Epi#37 T108, V112, L116, K115, Y119 T108, A110, L116, K115, N111 T13, V112, L116, K115, D118 P106, A110, L116, K115, N111 Epi#38 G64, E61, A40, V37, N27, P39, S38 G82, E102, A84, V99, N101, P106, S83 Epi#39 A110, E114, T108, P106, G122, L116 Epi#40 G14, G12, T17, K103, S83 R56, A52, T53, A54, S57 R66, A63, T65, K81, S83 R56, A72, T53, A54, S57 R56, G59, T53, A54, S57 R66, G64, Y67, K81, S83 Epi#42 P106, S83, G82, R75, R71 Epi#44 S1, Q3, D7, W2, Y5, S32, G12, T8 S1, Q3, D7, W2, Y5, A30, A36, P39 S1, Q3, D7, W2, Y5, S32, V11, T8 S1, Q3, D7, W2, Y5, S32, G12, T4 S1, Q3, D7, W2, Y5, A30, A33, T31 S1, Q3, D7, W2, Y5, A30, A36, T28 S1, Q3, D7, W2, Y5, S32, G12, T13 S1, Q3, D7, W2, Y5, S32, G34, T31 Epi#45 K93, H24, F49, R75, D74, G82 D25, H24, F49, R75, D74, G82 Epi#47 A36, G64, E61, A40, A44, A54, N58, R56, S57 Epi#50 D7, Y5, W2, T8, S1 D7, W2, W29, T28, P39 Epi#51 K90, H24, K93, D25, P39, T28, W29 T91, H24, K93, D25, P39, T28, W29 Profillin-Brich Pollen: Epi#01 L124, N118, P114, K88, K73, H68, Y74, R86, T95 Epi#02 T113, N118, Q116, P114, R86, V76 T50, K54, L62, T65, Y74, R86, V84 Epi#03 L133, K98, I129, Y127 Epi#04 S40, Q43, K45, T50, G32 S40, Q43, K45, T50, G51 S40, Q43, K45, T50, I49 Epi#05 G82, A81, A83, P59, G60, A63, T65, V76 G82, A83, A81, P59, G60, A63, H61, V76 G79, A81, A83, P59, G60, A63, T65, V76 Epi#06 G70, P46, D31, T50, K54, P59 A81, P59, D55, T50, Q47, P46 G32, P46, D31, T50, K45, P42 G51, P46, D31, T50, K54, P59 Epi#08 A81, E57, G60, A63, H61, F56 A81, E57, G60, V76, H68, F44 K54, E57, G60, A63, H61, F56 Epi#11 F56, K98, I85, Q78, V84, E122 F56, K98, I27, Q37, V34, D31 F56, K97, I85, Q78, V84, E80 Epi#12 Y6, E9 Y127, E122 Epi#13 H68, L62, P64, T65, A63, P59, A81, G82, G79 H61, L62, P64, T65, A63, P59, A81, G79, F56 H68, L62, P64, T65, A63, P59, A83, G79, G60 Epi#15 K45, P46, D31, I49, Q47, G32 K45, P46, D31, I49, K54, G60 K45, P46, D31, I49, K54, G82 K45, P46, D31, I49, T50, G51 Epi#16 Q116, P114, Y108, M12, S39, S40, A23, A24, D8 Q116, P114, Y108, M12, Q37, S40, A23, A24, D8 R86, P114, Y108, M12, S39, S40, A23, A24, D8 Epi#22 D126, L133, D130, Y127, E122 D130, L124, D126, Y127, E122 D130, L128, D126, Y127, E122 Epi#23 R123, N118, E122, L124, L11, A23 R123, N118, E122, L124, L11, A36 R123, N118, E122, L124, L11, A24 Epi#24 E109, G90, E110, P114, R86, E80, Q78 E57, K54, E58, P59, F56, A81, Q78 E58, G60, E57, P59, F56, E80, Q78 Epi#25 R86, K88, I107, E109, E110 R86, K88, I77, E80, V84 R86, K88, I107, E109, V112 Epi#27 57, E58, D55, K54 D55, E57, E58, K54 Epi#28 V34, D31, Q101, K98, E122, L128, Q131, G132, D130 I129, D126, Q131, L128, E122, K98, Q101, G100, D130 I72, H68, Q47, F44, E48, K45, Q43, G70, K73 I72, H68, Q47, I49, E48, K45, Q43, G71, K73 Epi#29 I129, Q101, L128, R123, E122 G132, Q131, L128, R123, E122 Epi#30 I77, M75, A63, H61, P59, L62, P64 G90, M75, A63, H61, K54, L62, P64 Epi#33 Q116, Y108, P111, S91, K89 K88, Y108, P111, S91, K8 Epi#34 V76, P64, M75, L62, G51, T50, P46 I27, W35, S33, V34, G32, T50, P46 V76, P64, T65, L62, G51, T50, P46 Epi#35 A24, L22, A23, S39, M12, 1107 A23, L11, A36, S39, M12, I10 Epi#37 Y127, R123, L124, K97, N118 Y108, A23, L11, R123, Y127 A23, A24, L11, R123, Y127 Epi#39 A81, E57, H61, T65, P64, G60, L62 A81, E58, H61, T65, P64, G60, L62 Epi#40 R123, L11, A23, Y108, P111, S91 R123, L11, A24, Y108, P111, T113 Epi#41 P111, Y108, L22, V112, S91 P114, Y108, L22, V112, S91 Epi#43 I27, W35, A36, L11, Q37, S39, M12, 1107, T95 Epi#44 I77, R86, P114, Y108, S91, V112, P111 V120, Q116, P114, Y108, S91, V112, P111 L22, Q116, P114, Y108, S91, V112, T113 L22, Q116, P114, Y108, A23, V112, P111 Epi#47 I129, Y127, E122, M119, R123, L124, N118, R86, P114 L133, Y127, E122, M119, R123, L124, N118, R86, P114 Epi#48 E122, Q116, P114, P111, V112 S91, K88, P114, P111, V112 Epi#50 H10, Y6, W3, S2, T5 H10, Y6, W3, T5, S39 Epi#51 K73, H68, K45, Q47, P46, S33, W35 Q101, H30, D31, T50, K45, Q47, H68 Rag Weed Pollen5: Epi#03 L4, K37, A33, I34, Y17 L4, K37, A33, I34, Y29 Epi#05 A33, N36, T40, G3, S20, L4 A33, N38, T40, G3, S20, Y25 A33, N36, G3, T40, S20, I22 Epi#06 A33, N36, D2, C19, K24, P21 A33, N38, D2, S20, K24, P21 Epi#09 I22, L4, D2, N38, D1, K37, A33, N36, T40 T9, G15, E7, V14, D30, K32, N36, T40, L4 T9, G15, E7, V14, D30, K32, N38, N36, L4 Epi#12 Y17, E7 Y6, E7 Epi#20 V27, K24, P21, L4, K37, D2, G3, D1 V27, K24, P21, L4, N36, D2, G3, D1 Epi#22 D1, D2, L4, K37 D1, D2, P21, K24 D2, L4, T40, D1 Epi#23 N10, E7, Y6, L4, P21 Epi#25 K32, I34, D30, V14 K37, I34, D30, V14 K16, I34, D30, V14 Epi#33 K32, Y17, V27, S20, K24 K16, Y6, P21, S20, K24 Epi#34 I22, P21, S20, V27, G12, Y17, T9 I22, P21, S20, V27, G12, Y29, S31 Epi#40 G12, G15, Y29, K37, T40 G15, G12, Y17, K16, T9 G12, G15, Y29, K32, S31 Epi#41 P21, Y6, L4, S20 Epi#44 L4, D2, P21, Y25, S20, V27, T40 L4, D2, P21, Y25, S20, G3, T40 Vesv5: Epi#01 L59, P67, P65, K143, K144, N64, Y140, R62, T61 L59, P67, P70, R57, K204, N73, Y201, Q202, T203 L59, P67, P69, R57, K72, N73, Y201, Q202, T203 L152, N149, P142, K145, K143, N64, Y140, R62, T61 Epi#02 L9, K7, Q108, P191, Y107, R102, V13 L9, K7, Q108, S192, Y107, R102, V13 Epi#03 L9, K7, A105, I6, Y3 Epi#04 K106, Y107, S192, Q108, K7, A105, I6 K106, Y107, S192, Q108, K7, V13, G12 Epi#05 G58, A56, R57, P69, G66, R62, T61, L59 G58, A56, R57, P69, G63, R62, T61, L59 Epi#06 G66, N64, D139, R62, K138, P67 G66, N64, D139, R62, K138, P65 G63, N64, D139, R62, K138, P67 Epi#08 K145, E199, S147, F151 K196, E198, S147, F151 K144, E199, S147, F151 Epi#09 L152, D150, S147, K144, N64, T61, L59 L152, D150, D139, K153, F151, S147, N197 D139, N64, R62, D135, K153, F151, S147, N197 Epi#10 E199, N197, N194, S147, F151, G148, K143 E199, N197, N194, S147, F151, G148, K196 E199, N197, N194, S147, F151, G148, K145 Epi#11 K179, I176, Q177, V30, E178 K29, I176, Q177, V30, E178 Epi#12 Y201, E199 Epi#13 S147, L200, P142, T203, A56, P70, L59, P67, G66 S147, L200, P142, T203, A56, P69, L59, P67, G58 S147, L200, P142, T203, A56, P70, L59, P67, G63 S147, L200, P142, T203, A56, P69, L59, P67, Y140 Epi#15 K106, P191, D103, I6, K5, A105 K106, P191, D103, I6, K7, G12 Epi#16 R57, P70, Y201, M74, Q53, N76, D50, A56, N73 R57, P69, Y201, M74, Q53, N76, D50, A56, N73 Q108, P191, Y107, R102, Q111, S192, D103, A105, N2 Epi#18 R57, L59, T61, P67, N64 R57, L59, T61, P65, N64 Epi#19 E167, N164, S192, Q108, R102, K7 E198, N194, S192, Q108, R102, K7 D103, T100, C8, Q108, R102, K7 Epi#22 L9, D103, T100, K10 A105, D103, L9, K7 D50, L45, D43, T37, K38 S147, D150, L152, K153 Epi#23 K196, N197, E199, N164, Q202, P70 K145, N197, E199, N164, Q202, P69 Epi#24 E198, K196, E199, P142, T203, P69, K143 E198, K145, E199, P142, T203, P70, K204 E198, K196, E199, P142, T203, P70, K72 E198, K145, E199, P142, F146, F151, K196 Epi#25 R57, K54, D50, N76 R57, K54, D50, E47 Epi#27 D43, E40, D125, K122 D50, E47, D43, K38 Epi#28 Q202, E199, K196, F151, S147, K144 Q202, E199, K196, F195, S147, K145 Epi#29 G58, R57, L59, R62, E136 G148, K145, L200, F195, E199 G148, K145, L200, F195, E198 Epi#33 K23, Y19, P24, S21, A16, K18 K23, Y34, P24, S21, A16, R102 Epi#34 I176, W180, T116, L115, G117, T119, S118 V31, P24, S21, L22, G35, Y34, T37 Epi#37 P69, R57, L59, K54, D50 P70, R57, L59, R62, D135 A56, R57, L59, R62, N64 P69, R57, L59, R62, D139 Epi#39 E199, L200, T203, P70, G58, L59 E198, L200, T203, P69, G58, L59 Epi#40 R57, L59, G58, T203, P69, T61 R57, L59, A56, Y201, K204, T203 R57, L59, A56, Y201, K72, T203 Epi#41 P24, Y19, L22, S21 P24, Y34, L36, S33 Epi#42 P191, S192, Q111, H98, R102, Q108 Epi#44 L59, R57, P70, Y201, A56, G58, T61 L59, R57, P69, Y201, A56, G58, T203 L59, R57, P70, Y201, A56, G58, P67 Epi#45 K153, H156, F151, Y140, N149, D150, L152 D135, H156, F151, Y140, N141, D150, L152 K143, P142, F146, Y140, N149, D150, L152 Epi#47 G58, L59, R57, M74, A56, Q202, N73, P70, P69 G148, Y140, R62, L59, R57, A56, N73, P70, P67 G66, G63, R62, L59, R57, A56, N73, P70, P67 G155, E136, R62, L59, R57, A56, N73, P70, P67 Epi#48 Q202, K204, P69, P67, G58 Q202, K204, P70, P67, G63 Q202, K72, P70, P67, G66 Epi#49 D125, D43, L45, V78, Q42, Q39, T37, K38 D125, D43, L45, V78, Q42, Q39, T37, K41 Epi#50 H98, Y96, W90, L22, S21 H98, Y96, W90, P24, S33 Epi#52 F0, A16, R102, W90, N25, Q95 F0, A16, R102, W90, N25, Q93 Betv1: Epi#03 SAS: 270, Size 11.07: L24, K20, H76, I23, Y81 SAS: 204, Size 11.96: L24, K20, A16, I23, Y81 Epi#05 SAS: 298, Size 14.01: G110, A106, A16, P14, G111, T10 SAS: 242, Size 14.01: G110, A106, A16, P14, G111, T107 Epi#08 SAS: 464, Size 11.12: K123, E127, G1, H121, F3 SAS: 455, Size 12.95: K129, E127, G1, H121, F3 SAS: 438, Size 13.31: K123, D125, G1, H121, F3 SAS: 428, Size 11.12: K123, E127, V2, H121, F3 SAS: 425, Size 11.65: K123, E127, G124, H121, F3 Epi#09 SAS: 466, Size 20.55: D109, A106, V105, K80, A16, T77 SAS: 444, Size 20.55: D109, G110, V105, K80, A16, T77 SAS: 427, Size 20.55: D109, G111, V105, K80, A16, T77 SAS: 398, Size 19.17: T10, G110, V105, K80, A16, T77 SAS: 381, Size 19.17: T10, G111, V105, K80, A16, T77 Epi#10 SAS: 558, Size 15.18: D75, T77, N78, A106, F79, R17, K20 SAS: 549, Size 21.96: E6, T7, N4, F3, G1, K123 SAS: 517, Size 13.31: D75, T77, N78, A16, F79, R17, K20 SAS: 497, Size 15.13: D75, T77, N78, A16, F22, R17, K20 Epi#12 SAS: 335, Size 9.08: T7, Y5, E6, N4 SAS: 331, Size 11.28: R145, Y150, E148, L152 SAS: 326, Size 10.37: R70, Y83, E73, P50 SAS: 311, Size 10.32: 1116, Y5, E6, N4 SAS: 308, Size 8.33: R145, Y150, E148, S149 Epi#18 SAS: 328, Size 24.67: S117, K103, F79, V105, A16, Y158, L24 Epi#22 SAS: 533, Size 9.96: D125, D93, K123, E127 SAS: 533, Size 9.96: D93, D125, K123, E127 SAS: 476, Size 11.40: D125, D93, K123, E96 SAS: 476, Size 11.40: D93, D125, K123, E96 SAS: 400, Size 17.99: D125, D93, P90, E87 Epi#23 SAS: 451, Size 22.02: K68, N43, E42, S57, F64, P63 SAS: 450, Size 22.02: K55, N43, E42, S57, F64, P63 SAS: 428, Size 22.02: K68, N43, E42, S57, L62, P63 SAS: 427, Size 22.02: K55, N43, E42, S57, L62, P63 SAS: 412, Size 18.85: K68, N43, E42, S40, F30, P35 Epi#24 SAS: 734, Size 18.92: E127, K123, E96, P90, S136, E131, K129 SAS: 729, Size 18.92: D93, K123, E96, P90, S136, E131, K129 SAS: 716, Size 19.57: E127, K123, E96, P90, S136, E131, K134 SAS: 711, Size 19.57: D93, K123, E96, P90, S136, E131, K134 SAS: 708, Size 20.49: D125, K123, E96, P90, S136, E131, K129 Epi#25 SAS: 467, Size 12.68: R70, K55, I44, E42, E45 SAS: 425, Size 12.68: R70, K54, I44, E42, E45 SAS: 420, Size 14.01: R70, K55, I44, D27, E42 Epi#27 SAS: 613, Size 14.25: D93, E127, A130, E131, K129 SAS: 595, Size 16.54: D93, E127, A130, E131, K134 SAS: 592, Size 16.70: D125, E127, A130, E131, K129 SAS: 574, Size 19.79: D125, E127, A130, E131, K134 SAS: 524, Size 18.78: D93, E127, A130, E131, K137 Epi#28 SAS: 869, Size 21.93: V33, Q36, F58, E60, L62, F64, P63, K65 SAS: 837, Size 21.83: V33, Q36, F58, E60, L62, F64, G61, K65 SAS: 808, Size 24.56: V33, Q36, F58, E60, L62, F64, P90, K65 SAS: 783, Size 21.83: V33, Q36, F58, E60, K65, F64, S57, K68 SAS: 782, Size 21.83: V33, Q36, F58, E60, L62, F64, S57, K65 Epi#29 SAS: 516, Size 9.52: G61, K65, L62, E60 SAS: 440, Size 8.70: G61, P63, L62, E60 SAS: 371, Size 6.78: G61, P59, L62, E60 Epi#32 SAS: 374, Size 17.88: F79, A16, A106, D109, V12 SAS: 354, Size 20.42: F22, A16, A106, D109, V12 Epi#33 SAS: 541, Size 18.79: K65, F64, P90, S136, A135, K134 SAS: 498, Size 9.15: Q36, F30, P35, S39, K32 SAS: 496, Size 11.27: Q36, F30, P35, S40, K32 SAS: 494, Size 12.19: Q36, F58, P35, S39, K32 SAS: 493, Size 18.79: K65, Y66, P90, S136, A135, K134 Epi#36 SAS: 447, Size 19.17: T77, A16, A106, V12, G110, T10 SAS: 430, Size 19.17: T77, A16, A106, V12, G111, T10 SAS: 392, Size 19.17: T77, A16, A106, V105, G110, T10 SAS: 391, Size 19.17: T77, A16, A106, V12, G110, T107 SAS: 375, Size 19.17: T77, A16, A106, V105, G111, T10 Epi#40 SAS: 246, Size 21.55: A106, A16, Y158, S155 SAS: 223, Size 13.25: A135, A130, Y5, T7 SAS: 196, Size 14.88: A135, A130, Y5, S117 SAS: 178, Size 10.62: A135, G140, T142, S136 Epi#44 SAS: 530, Size 19.04: L24, R17, D156, Y150, S149, V12, T10 SAS: 492, Size 19.04: 123, R17, D156, Y150, S149, V12, T10 SAS: 490, Size 17.39: L24, R17, D156, Y150, S149, V12, P14 SAS: 483, Size 23.09: L24, R17, D156, Y158, A16, A106, P108 SAS: 474, Size 20.83: L24, R17, D156, Y150, S149, V12, T107 Epi#45 SAS: 606, Size 21.41: K32, P35, F30, Y150, R145, V12 SAS: 546, Size 20.89: K32, P31, F30, Y150, R145, V12 SAS: 533, Size 15.19: K32, P35, F30, Y150, R145, G140 SAS: 533, Size 12.63: K32, P35, F30, Y150, R145, V33 SAS: 532, Size 19.60: K32, P35, F30, N28, D27, I44 Epi#47 SAS: 333, Size 21.03: R17, L24, N28, P31, P35 SAS: 300, Size 22.72: R17, L24, N28, P31, S39 SAS: 298, Size 21.80: R17, L24, N28, P31, S40 SAS: 269, Size 24.87: R17, L24, N28, P31, S57 Epi#48 SAS: 436, Size 14.26: S57, K65, P90, P63, G61 SAS: 414, Size 17.96: S39, K32, P35, P59, G61 SAS: 412, Size 17.96: S40, K32, P35, P59, G61 SAS: 389, Size 18.32: S57, K65, P63, P90, G92 SAS: 365, Size 21.15: S57, K65, P59, P35, V33

“SAS” is solvent accessible surface. “Size” is the total surface area of the epitope in A2.

Derf2: Epi#02 A98, K100, S101, P99, R128, R31 A98, K100, R128, P99, R31, V94 T91, N93, P95, P34, R31, R128 L61, N93, P95, P34, R31, R128 Epi#03 L40, K15, A39, I13, Y86 L40, K14, A39, I88, Y90 Epi#05 G32, A98, R31, P34, G20, T36, T91, Y90 G32, A98, R31, P34, G20, T36, T91, V94 G32, A98, R31, P34, G20, T36, T91, L37 G32, A98, R31, P34, G20, T36, T91, V18 Epi#06 A98, P99, D129, R31, K96, P95 G32, P99, D129, R128, R31, P95 A98, P99, D129, R31, K33, P95 A98, P99, D129, R31, K96, P34 A98, P99, D129, R128, K126, P26 Epi#07 T107, S57, D59, S101, R128, A98, P99, D129 T107, S57, D59, S101, R31, A98, P99, D129 Epi#08 K15, D87, V76, H74, F75 K14, D87, V76, H74, F75 K77, D87, V76, H74, F75 Epi#09 L61, D64, I68, H74, F75, T70, N71 N114, N46, D113, K48, N71, T70, T49 G83, N46, D113, K48, N71, T70, T49 Epi#10 L40, I13, D42, N44, V81, K48, N46, N114, G115 L40, I13, D42, N44, V81, K82, N46, N114, G115 L37, D19, G20, V18, V3, D4, K6, A120, T107, V105 Epi#11 F75, K51, I111, Q45, V116, D113 F75, K51, I111, Q45, V81, D113 Epi#12 Y90, E38 Epi#13 H30, R31, P95, A98, P99, S101, G60, L61 Epi#15 K96, P99, D129, 128, R128, A98 K96, P99, D129, I127, R128, A98

K96, P99, D129, 129, R128, A98

K55, P66, D64, I68, T70, G67 Epi#18 R31, R128, I28, G125, T123, H124, V105 R31, R128, I127, G125, T123, H124, V105 Epi#22 D1, M17, D4, V3, K6 D1, M17, D19, P34, K96 D1, M17, D4, V5, K6 Epi#23 K14, N11, E12, N44, Q85, P79 K14, N11, E12, N10, Q45, P79 K14, N11, E12, N44, Q84, P79 K14, N11, E12, L40, Q85, P79 Epi#24 D129, K100, E102, P99, R128, R31, K96 E62, G60, E102, P99, R128, R31, K96 D129, K126, E102, P99, R128, R31, K33 D129, K126, E102, P99, R31, P95, K96 Epi#25 R31, K96, I97, D59, E62 R128, R31, 197, D59, E102 R128, K126, I127, E102, N103 Epi#27 D64, E62, D59, K100 D59, E62, D64, K55 D87, E38, D19, K33 D19, E38, D87, K15 D19, E38, D87, K14 D19, E38, D87, K77 Epi#28 V16, D87, Q85, K14, E12, K15, Q2, D1 I13, D87, Q85, K14, E12, K15, Q2, D1 V3, D1, Q2, K15, E12, K14, Q85, D87 L40, D87, Q85, K14, E12, K15, Q2, D1 I88, D87, Q85, K14, E12, K15, Q2, D1 V76, D87, Q85, K14, E12, K15, Q2,D1 V18, D1, Q2, K15, E12, K14, Q85, D87 Epi#29 G32, N93, L61, E62 V94, N93, L61, E62 Epi#30 G60, I97, A98, H30, K96, P34, P95 I68, N71, H74, K77, P79, V81 G32, I97, A98, H30, K96, P95, P34 Epi#34 V105, P26, S24, G125, R128, S101, P99 W92, P34, T91, V94, R31, S101, P99 I28, P26, T123, G125, R128, S101, P99 Epi#37 A120, V16, L40, K14, N11 A39, V16, L40, K14, N11 Y90, A39, L40, K14, N11 Y86, A39, L40, K14, N11 Epi#39 A120, E38, T91, P34, G20, L37 A39, E38, T91, P34, G20, L37 Epi#40 G20, L37, A120, T123, K6, S24 A39, L37, A120, T123, K6, S24 G20, L37, A120, T107, K6, T123 Epi#41 P34, L37, V106, S57 Epi#42 P26, S24, G125, R128, R31 P99, S101, G125, R128, R31 Epi#44 V16, Q2, D19, P34, W92, Y90, A39, V18, T91 V16, Q2, D19, P34, W92, Y90, A39, V5, T123 V3, Q2, D19, P34, W92, Y90, A39, V18, T91 Epi#45 K77, H74, F75, N71, D69, G67 K77, H74, F75, N71, D69, V76 K77, H74, F75, N71, D69, V65 Epi#46 A98, R128, R31, P95, N93, G32 A98, R128, R31, P34, G20, Q2 Epi#48 Q2, D19, P34, P95, G32 H30, K96, P95, P34, G20 Epi#49 D87, D42, L40, Q85, Q84, C78, T47, Q45, K48 D87, D42, L40, Q85, Q84, C78, T47, Q45, K82 Epi#50 D19, W92, P34, T91 D19, W92, P34, P95 D19, W92, T91, T36 Epi#51 D129, H30, K33, R31, R128, K126, H124 R31, H30, D129, R128, K100, K126, H124 T123, H124, K126, R128, R31, K33, H30 Derp2: Epi#03 L17, K89, A39, I13, Y86 L17, K89, A72, I88, Y90 L17, K89, A72, I52, Y90 Epi#04 K15, S1, Q2, K14, V16, L17 K15, S1, Q2, K14, A39, L17 K15, S1, Q2, K14, V40, I13 Epi#05 G60, A56, L61, P99, G32, R31, H30, I97 G60, A56, L61, P99, G32, R31, H30, I28 Epi#06 G60, A56, D64, S57, K55, P66 G83, N46, D114, T49, K48, P79 G60, N103, D59, S101, R31, P95 Epi#08 K55, D64, S57, V106, F35 K55, E62, S57, V106, F35 Epi#09 L61, G60, E102, R128, 128, K126, N103, T123, V105 L61, G60, E102, R128, 1127, K100, N103, T123, V105 L61, G60, E102, R128, 1127, H124, N103, T123, V105 Epi#10 SAS: 435, Size 24.47: D69, T91, N93, F35, G32, R31 SAS: 422, Size 20.74: E38, T91, N93, F35, G32, K96 Epi#11 K14, I13, Q85, V81, E42 K15, I13, Q85, V81, E42 K14, I13, Q85, V40, D87 Epi#12 Y86, E42 Y90, E53 Y90, E38 Epi#13 H30, A125, P26, T123, A122, P19, L37, P34, W92 H30, A125, P26, T123, A122, H124, S24, G23, G20 H30, A125, P26, T123, A122, P19, L17, G20, F35 Epi#15 K55, P66, D69, I68, K89, A72 K55, P66, D69, I68, K89, A39 K55, P66, D64, I54, K109, G115 K55, P66, D64, I54, K109, A9 Epi#18 R31, 129, A125, S101, E102, N103 R31, 129, A125, S101, E102, V104 R31, 129, A125, T123, A122, V105 Epi#22 D69, P66, D64, V65, K55 D64, P66, D69, T91, K89 D59, L61, D64, P66, W92 D59, L61, D64, V65, E62 D69, P66, D64, V65, E53 Epi#24 D64, K55, E62, P99, R31, P34, K96 E53, K55, E62, P99, R31, P95, K96 D64, K55, E62, P99, R31, A98, K96 Epi#25 R31, H30, I28, E102, N103 R128, K126, I127, E102, N103 R128, K126, I28, E102, V105 Epi#27 D64, E53, D69, K89 D69, E53, D64, K55 D59, E62, D64, K55 Epi#28 V40, D87, Q85, E42, Q84, G83, K82 G20, H22, Q2, L17, E38, L37, Q36, P34, K33 G20, H22, Q2, L17, E38, L37, F35, P34, K33 Epi#29 I97, K100, L61, E62 G60, N103, L61, E62 I127, N103, L61, E62 Epi#30 G60, N103, S101, H30, K96, I97, P95 G60, N103, A125, H30, K96, I97, P95 I28, I127, A125, H30, K96, I97, P95 Epi#33 Q36, F35, V106, S57, A56, K55 K33, F35, V106, S57, A56, K55 Epi#34 I28, P26, S24, G23, G20, T123, S57 I28, P26, S24, V3, G20, T123, T107 W92, P34, T91, V18, G20, T123, P26 Epi#37 P66, V63, L61, K100, N103 P95, A98, L61, K100, N103 P19, V18, L17, K89, D87 P19, V3, L17, K89, D87 T123, V104, L61, K100, N103 Epi#38 L61, G60, E102, A125, V105, N103, P99, S57 L61, G60, E62, A56, V105, N103, P99, S57 Epi#39 A125, E102, H124, T123, P26, G20, L17 Epi#40 G60, L61, A56, T107, K6, T123 A39, L17, G20, T123, P26, S24 G60, L61, A56, T107, K55, S57 G60, L61, A56, T123, K126, S101 Epi#41 P19, L17, V3, S1 P19, L17, V5, S24 Epi#44 V65, D64, P66, W92, Y90, A39, V18, P19 L61, D64, P66, W92, Y90, A39, V18, T91 Epi#45 R31, P34, F35, N93, V94 K96, P34, F35, N93, G32 Epi#47 I127, S101, R31, 197, A98, L61, N103, P99, P95 I28, S101, R31, 197, A98, L61, N103, P99, S57 Epi#48 H30, K96, P95, P99, G60 H30, K96, P34, P19, G20 H30, K96, P34, P19, V18 H30, K96, P34, P95, V94 H30, K96, P34, P19, V3 E38, K89, P70, P66, V65 H30, K96, P95, P34, G32 Q36, K89, P70, P66, V65 Epi#50 D69, Y90, W92, P66, P70 D69, Y90, W92, P34, P95 D69, Y90, W92, T91, P34 D69, Y90, W92, V94, P95 D69, Y90, W92, L37, P19 Epi#51 K126, H124, E102, R128, 128, R31, H30 T123, H124, K126, R128, 128, R31, H30 D4, H124, K126, R128, 128, R31, H30

PhIp2:

Epi#02 T87, K85, Q61, S38, R34, R67 T87, K85, Q61, P63, R34, V42 Epi#03 K10, A90, I88, Y86 K10, A18, I88, Y86 Epi#04 R34, S38, Q61, K85, T87, I88 R34, S38, Q61, K85, T87, A90 Epi#05 G47, A18, S12, T87, G89, T91, T5, V1 G73, A29, L69, T27, G50, T53, T45, V42 G11, A18, L20, T91, G89, A90, T87, I88 Epi#06 A93, P94, D79, R34, Q61, P59 A93, P94, D79, R34, Q61, P83 A93, P94, D80, R34, Q61, P59 A93, P94, D79, R34, Q61, P63 Epi#08 K10, E9, G11, A18, H16, F54 K46, E48, G47, A18, H16, F54 K10, E9, S12, A18, H16, F54 Epi#09 L69, T27, G73, N76, R67, V77, D79, R34, A43, T45, V42 L69, T27, A29, E30, R67, V77, D80, R34, A43, T45, V42 Epi#10 D55, A18, N13, S12, F54, G47, K46 T45, A18, N13, S56, F54, G47, K46 Epi#09 L60, S56, E57, D55, K15, N13, S12, G11 L60, S56, E57, D55, H16, F54, T45, T53 L60, S56, E57, D55, H16, F54, T45, G47 Epi#12 Y86, E84 Y23, E24 Epi#18 N76, R67, F78, V81, A93, Y92, T91, T5, P2, V1 Epi#19 D39, W41, S38, Q61, R34, G37 E40, W41, S38, Q61, R34, A43 Epi#22 D79, P94, D80, P83, K85 D79, P94, D80, P63, K85 Epi#23 K10, N13, E14, L60, Q61, P59 K10, N13, E14, L60, Q61, P83 K10, N13, E14, L60, Q61, P63 Epi#24 E58, K15, E57, P59, S56, E14, Q61 D55, K15, E57, P59, S56, E58, Q61 Epi#25 R34, R67, W41, D39, E40 Epi#26 S38, E40, W41, V42, E32, E30 S38, E40, W41, V42, A43, E32 Epi#27 E14, E57, E58, K15 D55, E14, E84, K85 Epi#28 G37, H36, Q61, K85, E84, L60, F54, A43, K46 G37, H36, Q61, K85, E84, L60, F54, S12, D55 G37, H36, Q61, K85, E84, L60, F54, S56, D55 G37, H36, Q61, K85, E84, L60, F54, A43, R67 G37, H36, Q61, K15, E57, L60, F54, A43, K46 G37, H36, Q61, K85, E84, L60, F54, S12, K15 G37, H36, Q61, K85, E84, L60, F54, S56, K15 G37, H36, Q61, K85, E84, L60, F54, A43, R34 G37, H36, Q61, K85, E84, L60, F54, A18, D55 Epi#29 G73, K72, L69, R67, E30 I88, N13, L60, F54, E57 G25, K72, L69, R67, E32 V77, K75, L69, R67, E30 G37, H36, L60, F54, E57 G37, Q61, L60, F54, E57 Epi#30 I88, N13, S12, H16, K15, P59, L60 I88, N13, S56, H16, K15, L60, P59 I88, N13, A18, H16, K15, P59, L60 Epi#33 K46, F54, V42, S56, K15 H16, F54, V42, S56, K15 Epi#34 V1, P2, T5, V4, P94, Y92, T87 V1, P2, T5, L20, G89, T91, T87 V81, P94, T5, V1, P2, Y92, T91 Epi#37 T27, A29, L69, K72, D26 A43, R67, L69, K75, N76 Epi#38 L20, G89, E9, A18, N13, P59, S56 Epi#40 G49, L20, G89, Y86, K85, T87 G49, L20, G89, T87, K10, S12 G49, L20, G89, T87, K10, T7 Epi#44 V77, R67, D79, P94, Y92, A93, V1, P2 L69, R67, D79, P94, Y92, A93, V1, T5 Epi#45 D79, P94, F78, N76, M74, L69 D80, P94, F78, R67, D79, V77 K3, P94, F78, N76, M74, G73 Epi#46 A43, R67, R34, P63, H36, Q61 V77, R67, R34, P63, H36, G37 L69, R67, R34, P63, G37, Q61 Epi#47 G37, E35, E40, A43, R34, L60, N13, P59, S56 V77, E32, E40, A43, R34, L60, N13, P59, S56 S38, G37, E40, A43, R34, L60, N13, P59, S56 Epi#48 E24, K3, P94, P2, V1 E84, D80, P94, P2, V1 Epi#50 D39, W41, A43, T45 D39, W41, V42, T45 Epi#51 D79, H36, E84, T87, K10, G11, H16 D39, H36, Q61, K85, P63, R34, W41 D79, H36, E40, D39, G37, R34, W41 Q61, H36, E84, T87, K10, G11, H16 Example 11

For this example a third-generation epitope sequences were determined for some additional enzymes and redetermined for all of the enzymes in example 1-3. New enzymes are AMG (AMG pdb), BPN″ (1sup.pdb), Esperase (structure see Appendix D), Natalase (structure modelling based on SP722), Amylase-AA560 (Structure modelling based on SP722), Protease A, Alcalase, Protease B, ProteaseC, ProteaseD, ProteaseE, Properase and Relase based on their sequences and structures. The structures of Protease B, Properase, Relase, Protease A, Alcalase, ProteaseC, ProteaseD and ProteaseE can be found by “Homology modelling” (see above) and computer modelling of the epiope patterns that had been assembled in our database (shown in Table 8). Furthermore, the epitope sequences were redetermined for CAREZYME, Laccase, PD498, Savinase, Amylase SP722, and Cellulase, according to the method.

The protein surface is scanned for epitope patterns matching the given “consensus” sequence of about 6-12 residues. First, residues on the protein surface that match the first residue of the consensus sequence are identified. Within a specified distance from each of these, residues on the protein surface that match the next residue of the consensus sequence are identified. This procedure is repeated for the remaining residues of the consensus sequence. The method is further described under the paragraph “Methods” above and the program can be found in Appendixes.

The critical parameters used in this screening included:

    • i) a maximal distance between the alpha-carbon atoms of subsequent amino acids,
    • ii) a minimal accessability of the amino acid of 20 Å2,
    • iii) the largest maximal distance between the most distinct amino acids should be less than 25 Å
    • iv) the best epitope were taken,
    • v) the homology with the epitope pattern of interest was 100%

In this way a number of potential epitopes are identified. The epitopes are sorted according to total surface accessible area, and certain entries removed:

    • 1) Epitopes that contain the same protein surface residue more than once. These are artefacts generated by the described algorithm.
    • 2) Epitopes which are “too big”, i.e. where a distance between any two residues in the epitope exceeds a given threshold.

The subtilisin sequences and positions mentioned in the following are not given in the BPN′ numeration but in the subtilisins own numeration (see the alignment as described above in Tables 1A and 1B).

The epitope sequences found were:

AMG: Epi#01 L104, P123, P107, R125, R122, N182, S184, Q172, T173 L104, P107, P123, R125, R122, N182, S184, Q172, S453 L104, P107, P123, R125, R122, N182, S184, Q172, T452 Epi#02 L234, R241, S240, F237, T173, Y175, R122, R125 L234, R241, S240, F237, T173, Y169, R125, R122 L234, R241, S240, F237, T173, Y175, R125, R54 Epi#03 L291, K404, I288, Y289 L66, K61, H254, I253, Y329 Epi#04 R122, Y175, S184, Q172, Y169, A454, I455 R122, Y175, S184, Q172, Y169, N171, A451 R125, Y175, S184, Q172, Y169, T452, A451 Epi#06 G31, A24, D25, S30, A27, P41 G146, N145, D144, T148, S149, P467 A471, N145, D144, T148, S149, P467 Epi#07 G294, T290, S405, D293, S287, R286, P307, D283 G294, T290, S287, D293, S296, R286, P307, D283 G207, T204, S200, D214, S209, R160, P157, D153 G294, T290, S405, D293, S287, R286, P307, D309 Epi#08 A27, D25, S30, V111, F49 A24, D25, S30, V111, F49 Epi#09 S149, T148, G146, N145, A471, R68, N69, T72, V470 S73, S76, T72, N69, R68, A471, N145, T148 Epi#10 D238, N182, N236, S240, F237, R241, K244 D238, T173, N182, S239, F237, R241, K244 Epi#11 F49, F109,191, Q85, E113 Epi#12 Y363, E342 Y311, E308 Y175, E180 Epi#13 S119, W120, P123, A102, P94, S92, G90, L98 S119, W120, P123, A102, P94, S92, G96, G90 Epi#15 K244, P307, D283, I288, T290, G294 R160, P157, D153, I154, T462, G90 R286, P307, D283, I288, T290, G294 Epi#16 L410, P46, Y48, R413, S397, S394, A392, A393, N395 R160, P157, Y458, G456, S211, S209, A205, A201, D214 Epi#17 A201, S209, R160, S459 A205, S209, R160, S459 Epi#19 D44, N45, S411, Q409, R413, L410 D47, N45, S411, Q409, R413, L410 Epi#20 K61, P434, L66, L423, N427, D65, G70, D71 Epi#22 D357, S356, D349, V346, D345 D349, S356, D357, A359, D345 D357, S356, D349, L348, D345 Epi#23 K404, N292, E299, S298, L295, A300 K404, N292, E299, S296, L295, A300 Epi#24 D336, K337, E259, P258, S431, L332, K378 D336, K337, E259, P258, S431, R429, K378 D336, K337, A261, P258, S436, E259, Q338 Epi#25 R125, R122, W120, E180, N182 R241, K244, E308, N313 Epi#26 W212, S200, E198, W437, V197, G438, E259 W212, S200, E198, W437, V197, A201, D214 Epi#27 D283, E280, D349, K352 D403, E408, D406, K404 D349, E280, D283, K244 D349, E280, D283, K279 Epi#28 L332, D336, Q338, K337, E259, C262, P272, D345 V374, D336, Q338, K337, E259, C262, P272, D345 G339, D336, Q338, K337, E259, C262, P272, D345 Epi#29 L295, G294, L291, R286, E299 I288, K404, L291, R286, E299 L348, K352, L354, F380, E299 Epi#33 K352, Y355, V374, S371, S365, K337 K352, Y355, V374, S365, S340, K337 Epi#34 V463, W466, S468, V470, P467, T464, T462 I469, W466, S468, V470, P467, T464, T462 I154, W466, S468, V470, P467, T464, T462 V463, W466, S468, V470, P467, S465, T464 Epi#37 T362, A359, L348, K352, D357 T360, V346, L348, K352, D357 T362, A359, L348, K352, D349 Epi#38 G438, E259, A435, R68, L66, N69, P434, S431 Epi#39 A353, E299, R286, P307, G243, L234 A300, E299, R286, P307, G243, L234 Epi#40 A205, L143, G146, Y147, P467, T464 G146, L143, A205, T204, A201, S209 A451, A450, T448, P446, S444 Epi#41 P467, Y147, L143, V206, S149 Epi#42 L66, P434, S431, N430, R429, R428 L104, P123, S95, G101, P94, R122, R125 L104, P107, S95, G96, P123, R125, Q172 Epi#44 L143, Q140, D144, W141, Y147, S468, V470, T72 V206, Q140, D144, W141, Y147, S468, V470, P467 S211, Q216, D214, P218, Y223, A451, A450, T448 S211, Q216, D214, P218, Y223, A450, G447, T448 Epi#45 R413, P46, F49, Y50, N110, D112, G31 R413, P41, F49, Y50, N110, D33, G31 D44, P46, F49, Y50, N110, D112, G31 Epi#46 Y175, R125, R122, P123, G174, Q172 Y169, R125, R122, P123, G174, Q172 V432, R429, R428, P434, N69, G70 Y175, R125, R122, P94, N93, G90 Y175, R122, R125, P123, N182, G121 Y175, R125, R122, P94, G101, A102 Y175, R125, R122, P94, G118, A115 Y175, R125, R122, P94, G101, G96 Y175, R122, R125, P123, N182, G183 Epi#48 S211, D214, P218, P446, G447 E259, K337, P258, P434, V432 S215, D214, P218, P446, G447 S209, D214, P218, P446, V445 E259, K337, P258, P434, V433 Epi#50 R122, Y175, W120, T117, S119 R125, Y175, W120, S119, T117 Epi#51 T390, H391, E408, Q409, R413, S411, W317 T390, H391, E408, S405, I288, K404, W317 D406, H391, E408, Q409, R413, S411, W317 T390, H391, E408, D406, K404, Q409, W317 Epi#52 W437, A260, T266, R273, W228, D264, Q225 BPN′: Epi#02 T255, K256, S260, F261, P194, Y262, R186, V203 L257, K256, S260, F261, P194, Y262, R186, V203 T253, K256, S260, F261, P194, Y262, R186, V203 Epi#03 K141, A137, I108, Y104 K136, A137, I108, Y104 K136, A134, I108, Y104 Epi#04 K265, Y262, S188, Q185, R186, N184, L257 K265, Y262, S188, Q185, Y263, R186, L257 K265, Y262, S188, Q185, R186, N184, G258 K265, Y262, S188, Q185, Y263, R186, G258 Epi#05 G80, A1, N77, P40, G211, S38, S37, V44 G80, A1, N77, P40, G211, S38, S37, L42 G127, A152, N155, T164, G160, S158, S188, Y262 Epi#06 G211, N212, D36, S37, K43, P40 G80, N212, D36, S38, K43, P40 G211, N212, D36, S38, K43, P86 Epi#08 K256, D259, S260, F261 K43, D36, S38, V44, F58 Epi#09 S105, S132, A133, A137, D140, K141, A144, S145, N118 S248, T244, A144, S145, D120, K27, N118, A116, N117 Epi#10 E54, T55, N57, S37, F58, G46, K43 T55, A48, N57, S37, F58, G46, K43 E54, T55, N57, S49, F58, G46, K43 Epi#11 K136, I108, Q103, V51, D98 Epi#12 Y171, E195 Epi#13 S101, W106, P52, T55, A48, P56, S49, G47, F58 S105, W106, P52, T55, A48, P56, S49, G47, W113 Epi#15 N25, P239, D120, I115, K141, A144 N240, P239, D120, I115, K141, A144 Epi#16 Q271, P14, Y21, G20, Q19, S18, A15, A272, N252 Q59, P210, Y214, G211, S38, D36, D61, A99, D98 Epi#17 A187, S188, R186, S183 A187, S188, R186, S182 Epi#18 N184, R186, S188, G157, S158, T159, S161 N184, R186, S188, G157, S158, T159, S162 N184, R186, S188, G157, S158, E156, N155 N184, R186, S188, G157, S158, E156, F189 Epi19 E156, N155, S188, Q185, R186, L257 E156, N155, S188, Q185, R186, G258 E156, N155, S188, Q185, R186, A187 Epi#22 D197, S260, D259, L257, K256 D197, S260, D259, Y263, K256 Epi#23 N155, E156, S188, Q185, A187 Epi#24 E156, G166, E195, P194, S260, L257, K256 D259, G264, E195, P194, S260, L257, K256 D197, K170, E195, P194, S260, L257, K256 Epi#25 K141, I115, D120, N25 K141, I115, D120, N118 K141, I115, E112, N118 Epi#26 W113, S49, W106, P52, E54, D98 W113, S49, W106, P52, E54, D60 W113, S49, W106, V51, E54, D98 Epi#28 A99, D61, Q59, F58, E54, L96, Q103, G102, D98 A99, D98, Q59, F58, E54, L96, Q103, G100, D61 A99, D61, Q59, F58, E54, L96, Q103, S101, D98 Epi#29 G102, Q103, L96, E54 G100, Q103, L96, E54 Epi#30 I79, N76, S87, H17, S18, P14, V4 I79, N76, S87, H17, Q19, P14, V4 Epi#31 L257, Q185, N184, R186, F189, V203, I205, D181 L267, Q10, N184, R186, F189, V203, I205, D181 Epi#33 K213, Y214, P210, S38, S37, K43 Q59, F58, V44, S38, S37, K43 Epi#34 W106, P52, M50, G47, P56, T55, S53 W106, P52, S49, G47, P56, T55, S53 I115, W113, M50, V51, P52, T55, S53 I108, W106, S105, V51, P52, T55, S53 Epi#35 A99, L96, S49, M50, 1108 A99, L96, S49, M50, 1107 Epi#36 A137, A134, A133, G131, Y104, S105, Q103, V51, A48, W113 A134, A137, A133, G131, Y104, S101, Q103, V51, A48, W113 Epi#37 Y262, R186, L257, K256, D259 Y263, R186, L257, K256, N252 Epi#39 E156, T164, P129, G127, L126 E156, T164, P129, G128, L126 E156, T164, P129, G154, L126 E156, T164, P129, G166, L126 Epi#40 R247, L250, A272, T255, K256, S260 R186, L257, G258, Y263, K256, S260 G264, L257, G258, T255, K256, S260 Epi#41 P194, Y262, L257, S260 P194, Y263, L257, S260 Epi#42 P194, S260, G258, R186, Q185 Epi#44 S182, Q185, D181, Y6, S9, V4, P14 S183, Q185, D181, Y6, S3, V4, P5 S248, R247, D197, P194, Y262, S260, G258, T255 S53, P52, W106, Y104, S105, V51, T55 Epi#45 K170, P194, F261, Y262, R186, D181, V203 D197, P194, F261, Y262, R186, D181, V203 Epi#46 S162, S158, E156, N155, A187, Q185, N184, R186, S188 S188, S158, E156, N155, A187, Q185, N184, R186, S183 S158, S188, E156, N155, A187, Q185, N184, R186, S182 S161, S158, E156, N155, A187, Q185, N184, R186, S183 G160, S158, E156, N155, A187, Q185, N184, R186, S188 Epi#48 S38, K43, P40, P210, G211 S37, K43, P86, P14, V4 S38, K43, P40, P210, G215 Epi#50 H238, W241, T242, P239 H238, W241, T244, T242 H238, W241, T242, T244 Epi#51 T242, H238, Q275, Q271, P14, S18, H17 Q245, H238, Q275, K237, P239, T242, W241 Q275, H238, Q245, T242, R247, T244, W241 Q245, H238, Q275, Q271, P14, Q19, H17 CAREZYME Core: Epi#01 P61, P165, K164, R158, N154, Y168, R153, S151 P137, P49, K44, K13, N32, Y54, Q36, T39 P61, P165, K164, R158, N154, S152, R153, S151 Epi#02 L115, N118, S117, R4, T6, Y147, R146, V129 L115, N118, S5, R4, T6, Y147, R146, V129 Epi#03 K44, A43, I38, Y54 K13, A43, I38, Y54 Epi#04 R153, S151, Q145, Y147, R146, 1131 R153, S151, Q145, Y147, R146, G144 R153, S151, Q145, Y147, R146, L142 Epi#05 G3, A1, S183, T95, G101, A100, S96, G97 G3, A1, F184, T93, G101, T95, S96, G97 G97, A100, S96, T95, G101, T93, S183, G3 Epi#06 G140, P160, D161, R158, K164, P165 G50, P137, D133, R146, Q145, P143 A162, P165, D161, R158, K164, P160 Epi#07 G148, T6, S181, D178, R170, P165, D58 G128, T6, S181, D178, R170, P165, D58 Epi#08 K44, D42, S45, A43, F41 Epi#09 A191, E192, R196, A195, R200, N25, N202, N2O6 D161, R158, D157, R153, N176, S151, N154 Epi#10 D161, A57, N34, A162, F159, R158, K164 D2, A1, R185, S183, F184, G3, R4 Epi#11 F41, F29, I38, Q36, D58 Epi#12 Y168, E155 Y90, E91 Epi#13 A63, W62, P165, T60, A162, P160, L142, G149, Y147 A63, W62, P165, T60, A162, P160, L142, G128, Y147 A63, W169, P165, T60, A162, P160, L142, G144, Y147 Epi#15 P137, D133, I131, R146, G144 P137, D133, I131, R146, G148 P137, D133, I131, R146, G130 P137, D133, I131, R146, G128 P137, D133, I131, R146, G149 Epi#16 Q138, P137, Y54, R37, Q36, N34, A162, A57, D161 R170, P165, Y168, R153, S151, N176, D172, A63, D67 R170, P165, Y168, R153, S151, N176, D172, A63, D66 Epi#17 A1, S183, R4, S117 A100, S181, R4, S183 A1, S183, R4, S5 Epi#18 N118, R4, S181, - - - , G3, - - - , S117, L115, - - - , A78, S80 N34, N32, R37, F35, - - - , A33, Y54, S45, - - - , - - - , A43, V52 Epi#19 D157, N154, S151, Q145, R146, L142 D178, N176, S151, Q145, R146, G144 Epi#22 D40, A43, D42, W18, K20 D40, A43, D42, A19, K20 Epi#23 R158, N154, E155, L142, Q145, P143 R153, N154, E155, S151, Q145, P143 Epi#24 D42, K44, E48, P137, F139, A33, Q36 D40, K44, E48, P137, F139, A33, Q36 D161, K164, A162, P160, R158, L142, Q145 D161, K164, E155, P143, R158, L142, Q145 Epi#25 R158, K164, W169, D172, N176 R4, H119, 177, E82, N81 Epi#26 W18, S15, E82, W85, P23, A19, D42 W18, S15, E82, W85, P23, G84, D203 Epi#28 I31, D133, Q138, L142, E155, K164, F159, P165, D161 I131, D133, Q138, L142, E155, K164, F159, P143, R158 I131, D133, Q138, L142, E155, K164, F159, P160, R158 Epi#29 I131, R146, L142, R158, E155 G144, Q145, L142, R158, E155 Epi#30 G79, N81, A78, H119, S117, I77, L115 G79, N81, A78, H119, S76, I77, L115 Epi#31 L142, R158, N154, R153, W169, F171, D172 Epi#33 Q36, F29, P27, S15, A19, K20 K44, F41, P27, S15, A19, K20 Epi#34 V129, P143, S151, G144, R146, Y147, T6 V129, P143, S151, G148, R146, Y147, T6 V129, P143, S151, G149, R146, Y147, T6 Epi#36 A83, A22, A19, S15, K13, V52, A43, W18 Epi#37 Y147, R146, L142, R158, D161 Y147, R146, L142, R158, N154 Y147, R146, L142, R158, D157 Epi#38 E155, R158, P160, G140, L142 E155, R158, P143, G144, L142 Epi#40 G79, L115, G113, T111, A74, T6 G79, L115, G113, T111, A74, S15 G79, L115, G113, T111, A74, S110 G116, L115, G113, T111, A74, T6 G79, L115, G113, T111, A74, S76 Epi#42 L142, P143, S151, G144, R146, Q145 L142, P143, S151, G148, R146, Q145 L142, P143, S151, G149, R146, Q145 Epi#44 L142, R158, D161, P165, W62, Y168, S152, G144, P143 I131, R146, D133, P137, Y54, A33, V52, P49 L142, R158, D161, P165, W62, Y168, S152, G149, P143 Epi#45 R185, P208, F207, N206, D203, V24 D67, P213, F68, N65, D66, V64 R185, P208, F207, N206, D204, G205 Epi#46 A195, R200, R201, P23, N202, G205 A191, R200, R201, P23, N202, G205 V24, R201, R200, P190, Q211, A209 Epi#47 A191, A195, E192, V194, R200, N202, R201, P23 A195, A191, E192, V194, R200, N25, R201, P23 A191, A195, R196, V194, R200, N202, R201, P23 Epi#48 E48, K44, P49, P137, V52 E48, K44, P49, P137, G50 E48, K44, P49, P137, G140 Epi#50 D172, Y168, W62, V64, P213 D42, W18, A43, T39 D67, W173, W62, V64, P213 D66, W173, W62, V64, P213 D42, W18, S45, P49 D172, W169, W62, V64, P213 Epi#51 R4, H119, D2, T95, P98, K175, W169 R4, H119, D2, R185, P208, Q186, W85 R4, H119, D2, T95, G97, K175, W173 Epi#52 W18, A22, R200, R201, W85, Q186 Esperase: Epi#01 N24, P239, R237, K235, N243, S240, Q245, T242 N24, P239, K235, R27, N117, Y91, R43, S87 N24, P239, R237, K235, N243, Y241, Q245, S240 Epi#02 T3, N76, L75, R43, S38, Y209, R213, V215 T3, N76, S87, R43, S38, Y209, R213, V215 T129, N166, Q161, R160, T156, Y192, R186, V203 Epi#03 R186, Y192, S261, Q161, R160, N155, G127 R186, Y192, S261, Q161, R160, N155, G157 R186, Y192, S261, Q161, R160, N155, L126 R186, Y192, S261, Q161, R160, T156, G162 R186, Y192, S261, Q161, R160, N155, A187 Epi#05 G102, A105, S133, T134, G131, R170, T129, Y167 G102, A105, S133, T134, G131, R170, T129, G127 G211, A37, R43, P40, G80, T3, S78, I79 Epi#06 G211, N61, D97, R98, S53, P55 G102, N99, D97, R98, S53, P55 G100, N99, D97, R98, S53, P55 Epi#07 211, T210, D60, S38, R43, P86, D89 Epi#08 A108, E136, S133, A105, F50 A108, E136, S132, A105, F50 A187, D181, S188, V203, F189 Epi#09 N212, G211, S38, H59, N61, N99, R98 S52, S53, R98, N99, N61, G211 Epi#10 T129, T156, N155, S188, F189, G157, R160 D181, N183, R186, S188, F189, G157, R160 T129, N166, N155, S188, F189, G157, R160 T129, T156, N155, S218, F189, G157, R160 D97, N99, N61, S57, F50, G102, R98 Epi#12 Y167, E136 Y192, E195 Y171, E136 Epi#13 S38, R43, P40, A37, H59, S57, P55, Y58 S38, R43, P40, A37, H59, S57, P55, F50 S38, R43, P40, A37, H59, S49, P55, Y58 Epi#15 N24, P86, D89, I44, R43, A45 N24, P86, D89, I44, R43, G46 N76, P86, D89, I44, R43, A45 N24, P86, D89, I44, R43, A37 Epi#16 Q161, P194, Y192, G157, R160, S188, D181, A187, N183 Q161, P194, Y192, R186, Q185, S188, D181, A187, N183 Q161, P194, Y192, G162, R160, S188, D181, A187, N155 Epi#17 A37, S38, R43, S87 Epi#18 N144, N140, R141, L137, S133, T134, E136, S132 N140, N144, R141, L137, S133, T134, A105, S103 N143, N144, R141, L137, S133, T134, E136, N140 Epi#19 I21, N18, Q15, Q275, R19, G20 I21, N18, Q15, Q275, R237, G20 E197, N265, S261, Q161, R160, G162 E197, N265, S261, Q161, R160, G157 I21, N18, Q15, Q275, R237, G25 Epi#23 R98, N61, E54, S53, F50, P55 R98, N61, E54, Y58, F50, P55 R98, N61, E54, S57, F50, P55 R98, N61, E54, S52, F50, A105 Epi#24 E195, G264, E197, P260, S261, P194, Q161 D89, G46, A48, P55, S52, F50, Q109 E197, G264, E195, P194, S261, L262, Q161 Epi#25 R98, H59, E54, N61 R98, H59, D60, N61 R43, H39, I44, D89, N24 R27, H120,1115, E112, N116 Epi#28 L104, Q109, I115, E112, W113, F50, S53, R98 A105, Q109, I115, E112, W113, F50, G102, R98 A108, Q109, I115, E112, W113, F50, S53, R98 V107, Q109, I115, E112, W113, F50, S53, R98 Epi#29 I147, N140, L137, R141, E136 G146, N140, L137, R141, E112 I115, N143, L137, R141, E136 G102, N99, L96, R98, E54 Epi#30 G211, N212, S38, H59, S57, I51, P55 G211, N61, S57, H59, S38, P40, L75 G211, N212, S38, H59, S49, I51, P55 G211, N212, S38, H59, P55, I51, L96 Epi#31 L257, Q185, N183, R186, F189, V203, D181 L262, Q185, N183, R186, F189, V203, D181 Epi#33 H59, Y58, P55, S52, S53, R98 Q109, F50, P55, S57, S53, R98 Q109, F50, P55, S49, S53, R98 Epi#34 I79, P40, S38, G211, R213, Y209, S216 I79, P40, S38, G211, R213, Y214, T210 I51, P55, S49, L96, R98, S53, S52 Epi#37 T134, A108, L137, R141, N144 Y256, A254, L257, R186, N183 A105, A108, L137, R141, N144 Epi#38 L257, G264, E195, L262, N265, P260, S259 L257, G264, E195, L262, N265, P260, S261 Epi#39 E195, R170, P194, G264, L257 E195, R170, P194, G264, L262 Epi#40 R141, L137, A108, T134, A105, S133 R43, L42, A37, Y58, P55, S52 R186, L257, A254, Y256, P260, S259 R186, L262, G258, Y256, P260, S259 R186, L257, G184, Y256, P260, S259 R141, L137, A108, T134, A105, S103 R186, L262, G264, Y256, P260, S259 R186, L257, A254, Y256, P260, S261 R186, L262, G258, Y256, P260, S261 R186, L257, G264, Y256, P260, S261 Epi#41 P260, Y256, L257, S259 Epi#42 L75, P86, S87, N24, P239, R237, Q275 L75, P86, S87, N24, P239, R237, R19 Epi#44 S53, R98, D97, Y58, S57, A48, P55 S53, R98, D97, Y58, S38, G211, T210 Epi#45 R19, H17, F22, N24, D89, G25 R43, P86, F22, N24, D89, G25 R272, H269, F10, N183, D181, V203 R272, H269, F10, N183, D181, G184 R43, P86, F22, N24, D89, G46 Epi#46 R19, R237, P239, N24, G20 R19, R237, P239, N24, G25 Epi#47 G162, Y192, R160, N155, A187, Q185, N183, R186, S188 G157, Y192, R160, N155, A187, Q185, N183, R186, S188 S261, Y192, R160, N155, A187, Q182, N183, R186, S188 L262, Y192, R160, N155, A187, Q182, N183, R186, S188 Epi#48 S261, Q161, P194, P260, G258 S261, Q161, P194, P260, G264 Epi#50 D181, W6, V4, T3 D181, W6, V203, S188 D181, W6, V4, S9 D181, W6, T3, P5 Epi#51 R98, H64, T210, R213, P40, S38, H59 R98, H64, T210, R213, G211, S38, H59 R19, H17, Q15, Q275, R272, Q252, H269 Laccase: Epi#02 A14, N15, S17, F21, P180, Y176, R266, V177 T22, N15, P18, F21, P180, Y176, R266, V177 A274, N275, A181, R175, P180, Y176, R266, V177 A24, N15, S17, F21, P180, Y176, R266, V177 T272, N275, A181, R175, P180, Y176, R266, V177 Epi#03 L184, K173, I186, Y256 Epi#04 R234, S211, Q261, K264, N267, G271 R234, S211, Q261, K264, R266, G268, R259, S211, Q302, R234, N299, A301 R259, S211, Q236, R234, N299, A301 Epi#05 G372, A371, L369, P350, G81, S349, S351, V352 G372, A371, L369, P350, G81, S351, S349, Y347 Epi#06 G286, N289, D291, T293, S295, P292 G214, P252, D254, T293, S295, P298 A288, N289, D291, T293, S295, P292 Epi#07 G214, T294, D291, R283, V253, P252, D254 G30, T12, D53, R59, A497, P89, D51 G30, T10, D51, R59, A497, P55, D53 Epi#08 A371, E348, S349, A346, F335 A14, D53, G90, A92, H91, F93 A181, E183, G20, V16, F21 A181, E183, G20, A182, F21 Epi#09 N41, A100, N43, V6, D42, R37, N4, T8, L94 N41, A100, N43, V6, D42, R37, N4, T8, N47 L369, N366, E376, R379, N472, A471, V474 Epi#10 E183, A181, N275, T272, F273, G268, R266 D129, N41, N43, A100, F69, G72, R71 E183, A181, N275, A274, F273, G271, K264 Epi#11 F93, L486, I489, Q485, V481, E482 Epi#12 Y490, E488 Y375, E376 Epi#13 N366, P370, D367, I358, Q363, A471 N366, P370, D367, I358, Q363, G361 R379, P378, D326, I319, T321, G323 R379, P378, D326, I319, T321, G318 R379, P378, D326, I319, T321, A324 Epi#15 N366, P370, D367, I358, Q363, A471 N366, P370, D367, I358, Q363, G361 R379, P378, D326, I319, T321, G323 R379, P378, D326, I319, T321, G318 R379, P378, D326, I319, T321, A324 Epi#16 R175, P180, Y176, R266, Q164, N267, D166, A163, D205 R283, P292, Y256, G214, Q251, D254, A285, A288, N289 R283, P292, Y256, G214, Q251, D254, D291, A290, N289 Epi#17 A306, S413, R409, S414 A411, S413, R409, S414 A306, S410, R409, S414 A411, S414, R409, S410 Epi#19 E216, N250, Q251, Q191, R283, G286 E190, N250, Q251, Q191, R283, A288 E216, N250, Q251, Q191, R283, A290 E190, N250, Q251, Q191, R283, A285 Epi#22 D491, P494, D492, P495, E496 D492, P494, D491, L493, E496 Epi#23 R339, N460, E348, S349, L369, A371 R339, N460, E348, S351, L369, P370 R339, N460, E348, S351, L369, A365 R339, N460, E348, S351, L369, P350 R283, N188, E190, N250, Q191, P252 Epi#24 D475, G72, A476, P445, R379, A471, Q363 D53, G90, A497, P495, T498, P55, Q501 D53, G90, A497, P495, S499, L58, Q501 Epi#25 R37, K40, D129, N130 R37, K40, D129, N41 Epi#27 E142, E139, D138, K194, E142, E139, D138, K193 Epi#28 L58, Q501, I500, E496, L493, P495, D492 G286, D254, Q191, K194, E190, K193, G192, D138 A288, D254, Q191, K193, E190, K194, G192, D138 G192, D248, Q191, K194, E139, L136, A135, D138 V253, D254, Q191, K193, E190, K194, G192, D138 A285, D254, Q191, K193, E190, K194, G192, D138 Epi#29 G390, Q332, L329, R330, E435 V374, N366, L369, E348 I500, P495, L493, E496 G344, Q332, L333, R330, E435 Epi#30 G412, N304, A306, H309, I312, P314, V419 I312, L311, A315, H309, P229, L136, P132 Epi#31 L329, Q332, N343, R330, F331, V386, D434 L333, Q332, N343, R330, F331, V386, D434 L58, Q501, N54, R59, F112, M459, F456, D205 L58, Q501, N54, R59, F112, M459, I454, D205 Epi#33 Q485, Y490, P494, S499, A497, R59 Q251, Y256, P292, S295, A296, R234 H153, F21, V16, S17, A182, K173 H153, F21, P18, S17, A182, K173 Epi#34 V431, P395, T432, G433, G412, T415, S414 V431, P388, T432, G412, G433, S414, T415 V419, P320, T321, G323, P322, Y416, S414 V431, P395, T432, G390, G433, S414, T415 Epi#35 A371, L369, A362, S360, M359, I358 G372, L369, A362, S360, M359, I358 A365, L369, A362, S360, M359, I358 Epi#36 A362, A471, A476, V474, G361, S360, Q357, P350, A371, A365 A290, A288, A285, V253, Y256, S295, A296, W257 A288, A285, A287, V253, Y256, S295, A296, W257 Epi#37 P132, A135, L136, K194, N250 A135, A134, L136, K194, D138 P298, A301, L303, R234, N299 Epi#38 L356, G81, E348, A371, V374, L369, N366, P370, S351 L356, G81, E348, A371, V374, L369, N366, P370, S349 Epi#39 A411, E435, T432, P395, G393, L392 A1, E142, L35, R37, P34, G30, L27 A389, E435, T432, P395, G394, L392 Epi#40 R330, L333, G390, T432, A411, S414 G393, L392, G394, T432, A411, S414 R330, L333, G390, T432, A411, T415 Epi#41 P370, L369, V352, S351 P350, L369, V352, S351 Epi#42 L392, P395, S428, G430, P388, R330, Q332 Epi#44 S360, Q363, D367, P370, Y347, A371, G372, T345 V253, Q191, D254, P292, W257, Y256, S295, A296, P298 S360, Q363, D367, P370, Y347, S349, V352, P350 V253, Q191, D254, P292, W257, Y256, S295, G214, P252 Epi#45 R409, P322, F418, Y416, N420, D313, V419 K423, P314, F418, Y416, N420, D313, V419 R175, P180, F21, Y176, R266, D166, G268 Epi#46 A296, R259, R234, P300, N299, A301 Y256, R259, R234, P300, N299, Q302 Epi#47 I212, S211, R234, L303, A301, N299, P300, P298 I212, S211, R234, V232, A301, N299, P300, P298 Epi#48 S158, Q160, P157, P155, V504 S499, Q501, P55, P155, V504 E488, Q485, P480, P479, V481 Epi#49 D367, L369, V352, P350, Q357, Q363, M359, N478 D367, L369, P370, P350, Q357, Q363, M359, N478 Epi#50 D291, Y256, W257, S295, P298 D254, Y256, W257, T293, S295 Epi#51 D307, H309, E228, T218, P229, T231, H230 R234, H215, E216, T231, P229, H230, H309 D248, H215, E216, T231, P229, H230, H309 Epi#52 F69, A100, T98, R71, W75, T73, Q70 F97, A100, T98, R71, W75, T73, Q70 Natalase: Epi#01 P344, P382, R387, R33, N32, S28, R31, T36 P344, P382, R387, R33, N29, S28, R31, T36 Epi#02 A87, N21, Q18, R24, S28, R31, R33 A87, K89, S83, R24, S28, R31, R33 Epi#03 L307, K305, H402, I404, Y398 L307, K305, H401, I404, Y398 L307, K305, A304, I404, Y398 Epi#04 R167, S166, Q168, R172, N171, I173 R177, Y131, S128, Q125, R123, N124, I127 Epi#05 G178, A180, N124, P120, G190, S187, H234, L195 G178, A180, N124, P120, G190, R123, S187, Y192 G178, A180, N124, P120, G190, S187, H234, Y192 Epi#06 A87, N21, D25, R24, Q18, P14 G145, N146, D150, T147, R144, P142 G143, N146, D150, T147, R144, P142 G450, N451, D447, T455, K452, P453 A87, N21, D25, R22, Q18, P14 G454, N451, D447, T455, K452, P453 A378, P382, D447, T455, K452, P453 Epi#07 G145, T147, D150, S149, R213, V208, P205, D201 Epi#08 K305, D400, A304, H402, F399 K305, D400, A304, H401, F399 Epi#09 S79, S83, D25, R22, R24, H86, N90, S28, R31 N439, A460, N459, V444, K478, N417, T413, T414 Epi#10 E254, N249, R248, T245, F239, R212, R213 E254, N249, R248, T245, F239, R241, K275 Epi#11 F169, I173, Q170, D162 L195, I173, Q170, D162 Epi#12 Y192, E188 Y357, E354 Epi#13 H12, L13, P369, A375, P374, S372, P330, W11 H12, L13, P369, A375, P374, S372, P330, L334 H12, L13, P369, A375, P374, S372, P330, G331 Epi#15 N451, P453, D447, I448, T449, A378 N451, P453, D447, I448, K452, G450 Epi#16 Q313, P316, Y357, R353, Q395, D397, D400, A304, N308 Q355, P316, Y357, G356, R353, D397, D400, A304, D302 Epi#17 A87, S83, R24, S28 A87, S28, R24, S83 Epi#18 R33, N32, R31, S28, G92, N90 Epi#19 D16, N50, S48, Q49, R72, G69 D25, N21, Q80, Q18, R24, A87 E82, T77, Q18, Q80, R72, G69 Epi#22 D461, A460, W463, W433 Epi#23 K478, N417, E410, N439, Q438, A460 K478, N417, E410, N439, Q438, A441 Epi#24 E332, G331, E335, P330, S372, A375, K379 D381, K379, A375, P369, S372, P374, K377 Epi#25 R154, K138, W136, D162, N171 R213, R212, W217, E216, N249 R154, K138, W136, E134, N112 R241, K236, W183, D203, E206 Epi#26 W163, S166, E134, W136, V161, E117, E126 W163, S166, E134, W136, V161, E117, D130 W163, S166, E134, W136, V161, E117, D162 Epi#27 D203, E206, D201, K236 E117, E126, D130, K175 D201, E206, D203, K179 E126, E117, D162, K175 Epi#28 L195, D162, Q168, W163, E134, W136, Q165, S166, R167 I173, D162, Q170, W163, E134, W136, Q165, S166, R167 V161, D162, Q170, W163, E134, W136, Q165, S166, R167 Epi#29 G331, P330, L334, F337, E335 G178, K175, L114, R177, E117 Epi#30 G450, N451, H446, K478, I448, P453 G454, N451, H446, K478, I448, P453 Epi#31 Q168, N171, R172, W163, M196, I173, D162 Q170, N171, R172, W163, V161, I173, D162 Epi#33 K377, Y366, P369, S372, A375, K379 K377, Y366, P374, S372, A375, K379 Epi#34 W433, W463, T457, V444, G454, T455, P453 W433, W463, T457, V456, G454, T455, P453 Epi#37 Y156, R177, L114, K175, D130 T132, R177, L114, K175, N124 Epi#38 G429, E431, N469, P428, S472 G430, E431, N469, P428, S472 Epi#39 E10, H12, T370, P330, G331, L334 E10, L13, T370, P330, G331, L334 Epi#40 A378, A375, Y366, P369, S372 R177, L114, G178, Y156, K138, T110 A375, A378, Y366, P369, T370 Epi#41 P369, L13, V52, S48 Epi#42 P316, S281, G356, R353, Q355 P316, S281, G356, R353, Q395 Epi#44 V208, R213, W217, Y148, S149, G145, P142 S28, R33, D381, Y365, A378, A375, P369 L13, D16, P14, W11, Y362, A375, V373, T370 S333, D327, P330, W11, Y362, A375, V373, P369 Epi#45 D108, P142, F65, Y60, N146, D150, G145 D140, P142, F65, Y60, N146, D150, G145 Epi#46 Y392, R387, R33, P382, G450, G454 Y392, R387, R33, P382, Q388, G3 Epi#47 S83, S79, E82, I85, R24, A87, N90, R31, S28 A250, G252, E254, N249, R248, F256, N279, R241, S238 Epi#48 S372, H371, P374, P369, V373 Epi#49 D51, W11, L13, V52, P14, Q18, Q80, T77, N21 D51, W11, L13, V52, P14, Q18, Q80, T77, K74 Epi#50 D461, Y435, W433, W463, T457 D400, Y398, W433, W463, T457 D397, Y435, W433, W463, T457 Epi#51 T394, H396, D397, D400, K305, H402, H401 T455, H446, K478, T457, G442, Q438, W463 Epi#52 W136, A109, E134, R167, W163, N171, Q170 W136, A109, E134, R167, W163, N171, Q168 PD498: Epi#02 T262, K258, S260, F266, T198, Y196, R168, V166 T262, K258, S260, F266, T264, Y196, R168, V166 T141, N139, Q171, F170, S167, Y196, R168, V166 Epi#03 L99, K51, A49, I53, Y56 L99, K51, A49, I53, Y43 Epi#04 R28, S331, Q333, K97, R50, 153 R28, S331, Q333, K97, R50, A49 Epi#05 G108, A106, N107, G110, S109, S111, 159 G110, A106, N107, G108, S109, S111, L112 G108, A106, N107, G110, S111, S117, Y121 G108, A106, N107, G110, S111, S109, G135 G110, A106, L68, P214, G217, S219, Y220 G108, A106, N107, G110, S111, S109, L134 Epi#06 G135, N163, D164, R168, S174, P176 G162, N165, D164, R168, S174, P176 A22, N274, D25, S2, S9, P6 G154, N152, D148, T142, K144, P176 A22, P21, D25, S2, S9, P6 G154, N152, D148, S145, K144, P176 Epi#07 29, T332, S331, D95, S240, R28, V26, P21, D25 G29, T332, S330, D95, S331, R28, V26, P21, D25 Epi#08 K258, D257, S260, F266 K190, D185, S192, V207, F193 Epi#09 N215, N44, R50, 153, K54, N64, N63, R61 N44, A49, R50, 153, K54, N63, N64, R61 Epi#10 D188, N187, R189, S260, F266, G263, K258 D185, N187, R189, S260, F266, G263, K258 Epi#12 Y268, E253 Epi#15 R50, P46, D82, I87, T83, G86 N215, P46, D82, I87, T83, G86 Epi#18 N216, N44, R50, 153, A49, P46, N215 N215, N44, R50, 153, A49, P46, N216 Epi#19 D95, T332, S240, Q241, R28, G29 D95, T332, S330, Q241, R28, G29 Epi#22 D185, S192, D164, Y196, K267 D105, S111, D113, T141, K144 Epi#24 D95, K51, A49, P46, R50, K97 Epi#25 R120, K153, W151, D148, N152 R189, K190, D188, N187 R189, K190, D185, N208 Epi#27 D201, E253, D257, K258 D257, E253, D201, K267 Epi#28 I259, D257, Q254, E253, K267, F266, S260, R189 I259, D257, Q254, E253, K267, F266, S260, K258 Epi#29 L68, G108, L134, F170, E137 G135, N163, L134, F170, E137 Epi#30 G110, N107, A106, H71, L68, L104, L112 G108, N107, A106, H71, L68, P214, V213 G110, N107, A106, H71, P214, L68, L104 G110, N107, A106, H71, L68, L104, L134 Epi#33 Q12, Y220, V207, S222, S192, R189 190, F193, V207, S222, S192, R189 Q16, Y13, V207, S222, S192, R189 Epi#34 V26, W1, T27, G29, R28, S331, T332 W1, P21, T27, V26, R278, Y279, T255 Epi#35 G135, L134, S225, M221, I209 G110, L134, S225, M221, I209 G108, L134, S225, M221, I209 G162, L134, S225, M221, I209 Epi#37 A49, V52, L99, K54, N63 SAS: 309, Size 17.16: Y121, A127, L99, K54, N63 SAS: 307, Size 13.09: Y43, V52, L99, K54, N63 Epi#40 R189, G261, Y268, K258, S260 R189, G261, Y268, K258, T262 Epi#42 P3, S2, Q16, P21, R28, Q241 Epi#43 W199, Y196, G162, Q171, S140, L112, I115, T142 Epi#44 S145, D148, P176, W199, Y196, S167, G162, T169 S174, D201, P176, W199, Y196, S167, G197, T198 Epi#47 S330, S331, R28, V26, A22, Q16, N17, P21, S2 G242, S240, R28, V26, A22, Q16, N17, P21, S2 G29, S331, R28, V26, A22, Q16, N17, P21, S2 Epi#48 S2, D25, P21, P3, G86 S9, Q16, P21, P3, G86 Epi#50 R168, Y196, W199, T264, T198 D164, Y196, W199, T264, S260 Savinase: L21, N18, P14, R19, K231, N232, S236, Q239, S234 L21, N18, P14, R19, K231, N232, S234, Q230, S24 L21, N18, P14, R19, K231, N232, S234, Q230, T22 Epi#02 T254, N255, A188, R164, S158, Y186, R180, V197 T249, N263, Q12, R10, P14, R19, R269 T249, N263, S9, R10, P14, R19, R269

Epi#03

K27, A86, I43, Y89 Epi#04 K229, S234, Q230, K231, R269, A266 K27, S24, Q230, K231, R269, A15 K231, S234, Q239, R241, N246, A248 Epi#05 G187, A188, N255, T254, G252, S250, T249, L251 G189, A188, N255, T254, G252, S250, T249, L261 Epi#06 G252, N179, D175, S182, S154, P127 A188, N255, D191, R164, S158, P127 A188, N255, D191, R164, S128, P127 Epi#08 A131, E134, S139, A106, F49 A166, E134, S139, A106, F49 Epi#09 S103, T132, A131, E134, A166, R164, N167, S142, R143 Epi#10 D175, N177, N179, S182, F183, G155, R180 D175, N212, N153, S182, F183, G155, R180 Epi#11 F49, L94, 1105, Q107, V102, E134 F49, K92, I105, Q107, V102, E134 Epi#12 Y161, E134 Y165, E134 Epi#13 S76, L73, P39, T207, A209, P204, S206, G205, Y208 S85, L73, P39, T207, A209, P204, S206, G205, Y203 Epi#16 R164, P127, Y161, G152, S158, N255, D191, A166, N167 R164, P129, Y161, G152, S158, N255, D191, A166, N138 Epi#17 A156, S158, R164, S128 A188, S158, R164, S126 Epi#18 N177, N179, R180, S182, G155, S154, A156, S158 N177, N178, R180, S182, G155, S154, N153, F183 Epi#19 D175, N179, S182, Q185, R180, L256 D175, N179, S182, Q185, R180, L251 I240, W235, S234, Q239, R241, K245 D175, N179, S182, Q185, R180, G252 Epi#23 R143, N114, E110, S139, Q135, A131 R143, N115, E110, N138, Q135, A131 Epi#24 D58, G59, E53, P51, F49, P54, Q57 D58, G59, E53, P51, S48, P54, Q57 D58, G59, E53, P54, S55, F49, Q107 Epi#25 R19, R269, E265, N18 R269, R19, E265, N18 Epi#28 V102, Q107, F49, E53, K92, Q57, G46, R44 A47, Q107, F49, E53, K92, Q57, G46, R44 V50, Q107, F49, E53, K92, Q57, G46, R44 Epi#29 I77, N74, L41, R44, E87 V4, N74, L41, R44, E87 G20, N18, L21, R19, E265 Epi#30 G59, N60, S97, H62, L94, P51, P54 G98, N60, S97, H62, L94, P51, P54 Epi#31 L256, R180, N178, R10, W6, V197, D175 L251, R180, N178, R10, W6, V197, D175 Epi#33 Q107, F49, P51, S48, S55, K92 Q107, F49, P54, S55, A47, K92 Epi#34 V102, P129, S128, G125, R164, Y161, P127 V102, P129, S126, G125, R164, S158, P127

Epi#37

T254, A188, L256, R180, N177 T254, A188, L256, R180, N179 Epi#38 L94, G59, E53, A96, N60, P204, S206 L94, G59, E53, A96, N60, P204, S36 Epi#39 A131, E134, L133, T132, P129, G125, L124 A166, E134, L133, T132, P129, G125, L124 Epi#40 R44, L41, G78, T207, P39, T37 R19, L21, G20, T22, K231, S234 R180, L256, G252, T254, A188, S158 Epi#41 P127, Y161, L133, V102, S99 P127, Y161, L133, V102, S103 P127, Y161, L133, V102, S101 P127, Y161, L133, V102, S126 Epi#42 L73, P84, S85, N74, H17, P14, R19, R269 L80, P5, S3, N74, H17, P14, R19, R269 L21, P84, S85, N74, H17, P14, R19, R269 Epi#43 105, W111, A47, G46, Q57, S36, L41, I43, T37 Epi#44 S126, R164, P127, Y161, S158, A188, T254 S128, R164, P129, Y161, S158, A188, T254 Epi#46 A15, R269, R19, P14, N18, G20 A266, R269, R19, P14, N18, A15 Epi#48 S55, Q57, P54, P51, G52 E53, Q57, P54, P51, G52 Epi#50 R10, W6, S3, S76 R241, W235, S234, P233 R10, W6, V4, S9

Epi#51

Q239, H243, T247, R269, R19, K231, W235 R19, H17, E265, R269, K231, S234, W235 Epi#52 A15, S9, R10, W6, N198, Q176 A15, S9, R10, W6, N198, Q200 Amylase SP722: Epi#02 T419, N423, P422, F396, T5, Y398, R393, R37 T419, N418, P422, F396, T5, Y398, R393, R37 Epi#03 L313, K311, H408, I410, Y404 L313, K311, H407, I410, Y404 Epi#04 R171, S170, Q172, R176, N175, I177 R181, Y135, S132, Q129, R127, N128, I131 Epi#05 G184, A186, N128, P124, G196, S193, H240, L201 G184, A186, N128, P124, G196, R127, S193, Y198 Epi#06 G147, N150, D154, T151, R148, P146 G149, N150, D154, T151, R148, P146 Epi#07 G149, T151, D154, S153, R219, V214, P211, D207 Epi#08 K311, D406, A310, H407, F405 K311, D308, A310, H408, F405 Epi#09 T461, R485, K484, N423, T419, N418 R485, K484, N423, T420, T419 Epi#10 E260, N255, R254, T251, F245, R218, R219 T419, N423, N395, T5, F396, R393, R37 E260, T257, N255, T251, F245, R218, R219 Epi#11 F173, I177, Q174, D166 L201, I177, Q174, D166 Epi#12 Y363, E360 Y398, E360 Y198, E194 Epi#13 H16, L17, P375, A381, P380, S378, P336, W15 H16, L17, P375, A381, P380, S378, P336, G337 H16, L17, P375, A381, P380, S378, P336, L340 Epi#15 N457, P459, D453, I454, K458, G456 K458, P459, D453, I454, T455, A384 N457, P459, D453, I454, K458, G460 Epi#16 Q319, P322, Y363, R359, Q401, D403, D406, A310, N314 Q319, P322, Y363, G362, R359, D403, D406, A310, N314 Q319, P322, Y363, R359, R415, D403, D406, A310, N314 Epi#17 A91, S32, R28, S87 A91, S87, R82, S83 Epi#18 R485, V450, G448, T463, T461, H452, V462 N126, N128, R127, G196, Y198, S193, N195, N125 N25, R26, R28, S87, I89, A91, H90, N94 Epi#19 D20, N54, S52, Q53, R76, G73 D20, N19, Q22, Q84, R76, G73 D29, N25, Q22, Q84, R28, A91 Epi#20 K385, P350, L355, L313, K311, D308, G305, D432 Epi#22 D183, A186, D209, W189, K242 D183, A186, D209, W189, E190 D183, A186, D209, P211, E212 D209, A186, D183, Y160, W159 D183, A186, D209, W187, W189 Epi#23 R415, N418, E416, N445, Q444, A466 K446, N445, E416, Y441, Q444, A466 Epi#24 D387, K385, A381, P375, S378, P380, K383 E341, G337, E338, P336, S378, A381, K385 D333, G337, E341, P336, S378, A381, K385 Epi#25 R485, H452, I454, E391, N36 R485, K484, I454, E391, N395 Epi#26 W167, S170, E138, W140, V117, G182, D183 W167, S170, E138, W140, V165, E121, D134 W167, S170, E138, W140, V165, E121, E130 Epi#27 E212, E216, D154, K156 E216, E212, D209, K242 Epi#28 L201, D166, Q172, W167, E138, W140, Q169, S170, R171 L201, D166, Q169, W140, E138, W167, F173, S170, R171 L201, D166, Q174, W167, E138, W140, Q169, S170, R171 Epi#29 V214, N215, L217, R219, E222 G96, H90, L228, R82, E86 V214, R219, L217, R218, E212 Epi#30 G456, N457, H452, K484, I454, P459 G362, M323, S287, H324, K320, P322, V318 G362, M323, S287, H321, K320, P322, V318 G460, N457, H452, K484, I454, P459 Epi#31 L217, R219, N215, R218, F245, V214, D248 L217, R219, N215, R218, F245, M208, D209 Epi#33 K383, Y372, P375, S378, A381, K385 K383, Y372, P380, S378, A381, K38 Epi#34 W439, W469, T463, V450, R485, T461, P459 W439, W469, T463, V462, R485, T461, P459 Epi#37 T251, R218, L217, R219, N215 P211, V214, L217, R219, N215 A256, R218, L217, R219, N215 Epi#38 G435, E437, N475, P434, S478 G436, E437, N475, P434, S478 Epi#39 E338, H16, T376, P336, G337, L340 E14, H16, T376, P336, G337, L340 Epi#40 A384, A381, Y372, P375, S378 A384, A381, Y372, P375, T376 Epi#41 P375, L17, V56, S52 Epi#42 S378, P380, Y372, A381, A384, P375 S378, P375, Y372, A381, A384, P388 S378, P375, Y372, A381, A384, T455 Epi#45 K72, P146, F69, Y64, R148, D154, G149 K311, H408, F405, N409, D432, G304 D406, H408, F405, N409, D432, G304 Epi#46 Y398, R393, R37, P388, Q394, G7 Y398, R359, R393, P388, G456, G460 Y398, R393, R37, P388, Q394, G38 Epi#47 A256, G258, E260, N255, R254, F262, N285, R247, S244 S193, Y198, E194, N125, R127, Q129, N123, R176, P124 Epi#48 S378, H377, P380, P375, V379 H16, H377, P375, P380, V379 Epi#49 D55, W15, L17, P18, Q22, Q84, T81, N25 D55, W15, L17, P18, Q22, Q84, T81, K78 Epi#50 D467, Y441, W439, W469, T463 D406, Y404, W439, W469, T463 D183, Y160, W159, W140, T114 D403, Y441, W439, W469, T463 Epi#51 D406, H408, D308, K311, L313, Q319, H321 Epi#52 W140, A113, E138, R171, W167, N175, Q174 W140, A113, E138, R171, W167, D166, Q172 Amylase AA560: Epi#01 L390, P388, P350, K383, K385, N457, S478, R458, T461 L390, P388, P350, K383, K385, N457, S478, R458, T452 L390, P388, P350, K383, K385, N457, S478, R458, T455 Epi#02 L390, K395, Q394, R393, T5, Y398, R359, R400 L173, K172, S170, T136, Y135, R118, R181 L173, R171, S170, T136, Y135, R118, R181 L390, K395, Q394, R393, T5, Y398, R400, R415 Epi#03 K438, H407, I410, Y404 Epi#04 K172, S170, Q169, R171, N174, L173 R171, S170, Q169, K172, N175, I177 Epi#05 G456, A459, R458, T461, G460, T452, T463, V450 G456, A459, R458, T452, G460, T461, T463, G448 Epi#06 A51, N54, D20, R76, Q71, P146 G73, A51, D55, S52, K72, P146 Epi#07 G456, T455, S384, D387, R393, P388, D453 Epi#08 K259, S255, V222, H252, F245 K259, G258, A256, H252, F245 Epi#09 N128, V131, R176, D166, K172, N175, N174, R171 Epi#10 467, N445, R444, F441, R415, R400 D467, A466, R444, F441, R415, R400 Epi#11 F69, K72, I75, Q53, V56, D55 Epi#12 Y16, E337 Y363, E360 Y198, E194 Epi#15 K385, P388, D453, I454, R458, A459 K385, P388, D387, I454, T452, A459 K385, P388, D387, I454, R458, G456 Epi#17 A87, S29, R28, S32 A91, S29, R28, S32 Epi#18 N445, R444, A466, T463, T461, N471, N437 N445, R444, A466, T463, T461, T452, V450 Epi#19 166, W167, S170, Q169, R171, K172 E138, W167, S170, Q169, R171, K172 E134, T136, S170, Q169, R171, K172 Epi#22 D209, P211, D207, Y160, D183 Epi#23 R400, N418, E416, N445, Q449, A466 R82, N83, E68, N70, F69, P146 Epi#24 E134, G133, E130, P124, R176, L173, K172 E134, K179, E130, P124, R176, L173, K172 Epi#25 R444, K446, W469, D467, N445 R171, K172, W167, D166, N175 R171, K172, W167, D166, N174 Epi#26 W167, S170, E138, W140, V165, E121, E130 W167, S170, E138, W140, V165, E121, E134 W167, S170, E138, W140, V165, E121, D166 Epi#27 E130, E121, D166, K172 D36, E391, D387, K385 E134, E121, D166, K172 Epi#28 L201, D166, Q169, W140, E138, K172, S170, R171 L173, D166, Q169, K172, E138, W167, S170, R171 Epi#29 V131, R176, L173, R171, E138 I177, N175, L173, R171, E138 I177, N174, L173, R171, E138 Epi#30 I39, N33, S29, H23, P18, L17, P375 G38, N33, S29, H23, L17, P375, P380 G362, M323, S287, H321, Q319, P322, V318 G417, N423, A420, H421, K395, L390, P388 G21, N25, S29, H23, P18, L17, P375 G399, N418, A420, H421, K395, L390, P388 Epi#31 L173, R171, N174, R176, W167, M202, I177, D166 L173, R171, N174, R176, W167, V165, I177, D166 Epi#33 K108, Y58, V56, S52, A51, K72 Epi#34 W439, W469, T463, V450, G460, T452, T461 W15, P18, T376, G378, P375, Y372, S384 W469, W439, S473, G460, R458, T461, T463 Epi#37 P124, R176, L173, K172, N175 P124, R176, L173, R171, N174 Epi#40 R400, G399, Y396, P422, T419 R400, G417, Y396, P422, T419

Epi#41

P375, Y16, L17, V56, S52 P18, Y16, L17, V56, S52 Epi#42 P350, S478, G433, H408, R310, Q311 P322, S287, N285, H324, R320, Q319 P322, S287, G362, H321, R320, Q319 Epi#44 L17, D20, P18, W15, Y368, A381, G378, T376 L340, D333, P336, W15, Y368, A381, G378, P375 Epi#45 K72, P146, F69, Y64, N150, D144, G147 D112, P146, F69, Y64, N150, D144, G149 Epi#46 Y398, R359, R393, P388, G456, A459 Y363, R359, R393, P388, Q394, G7 Y363, R359, R393, P388, Q394, G38 Epi#47 I75, E68, R76, N83, R82, Q84, N90, R28, S29 G133, E134, E130, V131, R176, L173, N174, R171, S170 Epi#48 S384, K383, P380, P375, G378 E337, H377, P380, P375, V379 Epi#50 R444, W469, W439, S473, T461 D183, Y160, W159, W140, T114 Epi#51 R320, H321, Q319, P322, H324, H286 Epi#52 W140, A113, E138, R171, W167, D166, Q169 W140, A113, E115, R118, W159, T114, Q169 Protease A: Epi#01 L21, N18, P14, R19, K237, N238, S242, Q245, S240 L21, N18, P14, R19, K237, N238, S240, Q236, S24 Epi#02 T255, N269, Q12, R10, P14, R19, R275 T255, N269, S9, R10, P14, R19, R275 Epi#03 K27, A88, I44, Y91 Epi#04 K235, S240, Q236, K237, R275, A15 K27, S24, Q236, K237, R275, A15 K237, S240, Q245, R247, N252, A254 R145, S141, Q137, Y171, N173, A172 Epi#06 G61, N62, D60, T38, Q59, P55 G211, P210, D60, T38, Q59, P55 A98, N62, D60, T38, Q59, P55 G100, N62, D60, T38, Q59, P55 Epi#08 A131, E136, S141, A108, F50 A172, E136, S141, A108, F50 A98, E54, G53, V51, F50 Epi#09 S162, S170, A172, N173, V244, H249, N252, S256, T260 S259, S256, T260, N261, L262, R186, N185, S188, N155 S162, S170, A172, N173, V244, H249, N248, N252, T255 S156, S162, N261, S259, L262, R186, N185, S188, N155 Epi#10 D181, N183, N185, S188, F189, G157, R186 D181, N218, N155, S156, F189, G157, R186 Epi#12 Y171, E136 Y91, E89 Epi#13 S78, L75, P40, T213, A215, P210, S212, G211, Y209 S87, L75, P40, T213, A215, P210, S212, G211, Y214 Epi#16 L262, P194, Y192, G195, S162, N261, D197, A172, N140 L262, P194, Y192, G157, S162, N261, D197, A172, N173 L262, P194, Y192, G161, S162, S170, D197, A172, N173 Epi#17 A138, S141, R145, S144 A108, S141, R145, S144 Epi#18 N185, N183, R186, L262, S259, T260, P194, N261 N185, N183, R186, L262, Y192, T260, P194, S162 Epi#19 I246, W241, S240, Q245, R247, K251 D181, N185, S188, Q191, R186, L262 Epi#23 R145, N116, E112, S141, Q137, A138 R145, N117, E112, S141, Q137, A108 Epi#24 E136, G133, A131, P129, S103, F50, Q109 E136, G132, A131, P129, S103, A108, Q137 D60, G61, E54, P52, F50, P55, Q59 Epi#25 R275, R19, E271, N18 Epi#28 G20, H17, Q12, E271, L21, Q236, S240, K237 A15, H17, Q12, E271, L21, Q236, S240, K237 Epi#29 V244, Q245, L148, R145, E112 V244, N173, L148, R145, E112 Epi#30 G61, N62, A98, H64, L96, P52, P55 G20, N18, A15, H17, S87, L75, P40 I79, N76, S87, H17, Q12, P14, V4 G100, N62, A98, H64, L96, P52, P55 Epi#31 L262, R186, N184, R10, W6, V203, D181 L257, R186, N184, R10, W6, V203, D181 Epi#33 Q109, F50, P52, S49, S56, K94 Q109, F50, P55, S56, A48, K94 Epi#34 W241, P239, S242, G146, R145, S141, S144 I165, P194, T260, G258, R186, S188, S156 V104, P129, S130, G127, G102, S101, S99 V244, W241, S242, G146, R145, S141, S144 I165, P194, S170, G127, P129, S130, S103 Epi#37 P14, A15, L21, R19, N18 T143, R145, L148, R247, N252 T143, V244, L148, R145, N116 Epi#38 L96, G97, E54, A98, N62, P210, S212 L96, G97, E54, A98, N62, P210, S37 Epi#39 A15, E271, H17, R19, P14, G20, L21 A254, E271, H17, R19, P14, G20, L21 A272, E271, H17, R19, P14, G20, L21 Epi#40 R186, L257, G258, T260, P194, S162 R186, L262, G161, Y192, P194, T260 Epi#41 P194, Y192, L262, S259 P194, Y192, L196, S162 Epi#42 L82, P5, S3, N76, H17, P14, R19, R275 L82, P5, S9, Q12, H17, P14, R19, R275 Epi#43 W113, A48, G47, Q59, S37, L42, I44, T38 Epi#44 V244, R247, D197, P194, Y192, S162, G195, T260 V244, R247, D197, P194, Y192, S170, G195, T260 S56, Q59, D60, P210, Y214, S212, G211, T38 S56, Q59, D60, P210, Y209, S212, G211, T38 Epi#46 A15, R275, R19, P14, N18, G20 A272, R275, R19, P14, N18, G20 A272, R275, R19, P14, N18, A15 Epi#47 S130, A131, E136, N173, A172, N140, R145, S144 S105, A131, E136, N173, A172, N140, R145, S144 Epi#48 E54, Q59, P55, P52, G53 S56, Q59, P55, P52, G53 S49, Q59, P55, P52, G53 Epi#50 R10, W6, S3, S78 R10, W6, V4, S9 R10, W6, V203, S188 Epi#51 Q245, H249, T253, R275, K237, S240, W241 R19, H17, E271, R275, K237, S240, W241 R145, H120, K27, S24, K237, S240, W241 R145, H120, K235, K237, P239, S240, W241 Epi#52 A15, S9, R10, W6, N204, Q206 A15, S9, R10, W6, N204, Q182 Alcalase: Epi#01 L10, P5, P9, K15, K12, N269, S251, R249, T253 L82, P5, P9, K15, K12, N269, S251, R249, T253 Epi#02 T115, N141, A144, R145, S242, R247, R249 A138, N141, A144, R145, S242, R247, R249 Epi#03 L196, K170, A129, 1165, Y167 L196, K170, A194, I165, Y171 Epi#04 R145, Y143, S173, Q137, K136, T133, A134 K170, Y167, S132, Q137, K136, N141, A144 Epi#05 G53, A52, F50, G102, S105, S103, Y104 G53, A52, F50, G102, S101, S103, Y104 Epi#06 A24, N25, D120, R145, S242, P239 A144, N141, D140, R145, S242, P239 Epi#08 K265, E197, S260, A194, F261 A56, E54, G53, A52, F50 Epi#10 T162, N161, N163, A194, F261, G264, K265 E195, N161, N163, S158, F261, G258, K265 Epi#12 Y57, E54 Y262, E197 Epi#13 S38, A37, P40, T213, A215, H64, L217, G204, Y206 S38, A37, P40, T213, A215, H64, S98, G100, G61 S87, L75, P40, T213, A215, H64, L217, G204, Y6 Epi#16 L10, P9, Y6, G204, S182, N183, D181, A187, N185 Q2, P5, Y206, G204, S182, N183, D181, A203, N218 L10, P9, Y6, G204, S182, N183, D181, A187, N155 Epi#17 A144, S244, R247, S252 A272, S252, R249, S244 A144, S244, R249, S251 A254, S252, R249, S244 Epi#18 N141, R145, A144, Y143, S244, N248, S252 Epi#19 N248, S244, Q245, R249, A272 N240, S242, Q245, R249, A254 N240, S242, Q245, R249, L241 Epi#22 D76, L82, D14, A18, K15 D181, L10, D14, A18, K15 Epi#23 K27, N117, E112, N141, Q137, A134 K27, N117, E112, N141, Q137, A138 K27, N117, E112, S109, F50, A52 Epi#24 D120, K27, A24, P86, F21, A18, K15 D14, K22, A24, P86, F21, A18, K15 D76, K22, A24, P86, S87, F21, K15 Epi#25 R249, R247, E197, E195 Epi#27 D172, E195, E197, K265 E197, E195, D172, K136 D172, E197, E195, K170 Epi#28 A18, D14, Q19, K15, E271, K12, Q17, S87, D76 V4, D14, Q17, K12, E271, K15, F21, A18, K22 Epi#29 L257, K265, L196, F261, E195 G53, N97, L96, F50, E54 Epi#30 G146, L241, S242, H238, K237, P239, L235 G146, L241, S236, H238, S242, P239, L235 Epi#33 K15, F21, P86, S87, A24, K27 K27, Y91, V45, S89, A24, K22 Epi#34 V4, P5, T3, G80, P40, S38, T211 V108, W113, T116, G118, R145, Y143, S244 V26, P239, S242, G146, R145, T115, T116 Epi#36 A52, A56, A48, V51, G102, Y104, S105, V108, A138, A134 A52, A56, A48, V51, G102, Y104, S103, V108, A134, A138 Epi#37 Y262, A194, L196, K265, Y256 Y263, R186, L257, K265, Y256 Y256, A254, L257, K265, Y262 Epi#40 R186, L257, A254, Y256, K265, S252 R186, L257, G258, Y256, K265, S260 Epi#41 Y256, L257, S260 Y256, L257, S259 Epi#42 L235, P239, S242, N248, R249, Q275 L241, P239, S242, Q245, R249, Q275 Epi#44 S132, Q137, D140, Y143, A144, A138, T133 V108, Q137, D140, Y143, A144, A138, T133 S173, Q137, D140, Y143, A144, A138, T133 Epi#48 Q19, K15, P9, P5, V4 E271, K15, P9, P5, V4 Protease B: Epi#05 SAS: 454, Size 24.86: G189, A188, R164, P127, G125, S99 SAS: 452, Size 15.92: G189, A188, R164, P127, G125, S128 SAS: 451, Size 24.86: G157, A188, R164, P127, G125, S99 SAS: 449, Size 15.92: G157, A188, R164, P127, G125, S128 SAS: 445, Size 23.31: G189, A166, R164, P127, G125, S99 Epi#09 SAS: 446, Size 15.76: T254, G189, A166, R164, A188, S158 SAS: 312, Size 15.90: T22, G20, L21, R19, A15, S9 Epi#10 SAS: 460, Size 17.32: D175, N177, N179, S182, F183, G155, R180 SAS: 437, Size 16.70: D211, N212, N153, S182, F183, G155, R180 SAS: 424, Size 13.75: D175, N212, N153, S182, F183, G155, R180 SAS: 417, Size 16.70: D211, N212, N153, S154, F183, G155, R180 SAS: 404, Size 15.83: D175, N212, N153, S154, F183, G155, R180 Epi#12 SAS: 309, Size 13.46: P127, Y161, E134, P129 SAS: 292, Size 9.37: R164, Y161, E134, P129 SAS: 287, Size 18.66: P127, Y161, E134, N138 SAS: 284, Size 16.85: P127, Y161, E134, N167 SAS: 275, Size 11.53: S128, Y161, E134, P129 Epi#17 SAS: 275, Size 15.84: A188, S158, R164, S126 SAS: 225, Size 12.79: A156, S158, R164, S126 Epi#18 SAS: 444, Size 16.32: S250, K245, S259, L256, A188, T254, L251 SAS: 397, Size 14.14: S250, K245, S259, L256, G252, T254, L251 SAS: 397, Size 14.14: S250, K245, S259, L251, G252, T254, L256 SAS: 397, Size 14.14: S259, K245, S250, L251, G252, T254, L256 SAS: 396, Size 21.52: S158, R164, S126, V102, G100, S99, L124 Epi#19 SAS: 295, Size 15.06: D175, W6, S9, Q12, R10 SAS: 278, Size 21.23: E110, T141, S236, Q239, R241 Epi#23 SAS: 486, Size 19.88: R143, N114, E110, S139, Q135, A131 SAS: 473, Size 18.68: R19, N18, E265, L21, Q230, P233 SAS: 468, Size 15.74: R164, N167, E134, S139, Q135, A131 SAS: 463, Size 13.77: R164, N167, E134, S130, Q135, A131 SAS: 461, Size 21.98: R44, N42, E87, S24, Q230, P233 Epi#28 SAS: 520, Size 19.27: V102, Q107, W111, E110, Q135, S139, R143 SAS: 492, Size 24.70: V102, Q107, F49, E53, Q57, G46, R44 SAS: 480, Size 22.76: V50, Q107, W111, E110, Q135, S139, R143 SAS: 452, Size 19.08: V50, Q107, F49, E53, Q57, G46, R44 SAS: 441, Size 24.70: V102, Q107, E110, W111, F49, G46, R44 Epi#29 SAS: 239, Size 11.49: G20, N18, L21, E265 SAS: 224, Size 11.49: G20, R19, L21, E265 SAS: 179, Size 16.62: 14, P14, L21, E265 SAS: 175, Size 11.49: G20, K231, L21, E265 SAS: 153, Size 18.96: G25, Q230, L21, E265 Epi#30 SAS: 308, Size 24.27: G20, L21, A15, H17, S85, L73, P39 Epi#31 SAS: 363, Size 21.72: L256, R180, N178, R10, W6, V197, D211 SAS: 352, Size 22.95: L251, R180, N178, R10, W6, V197, D211 SAS: 350, Size 21.62: L256, R180, N178, R10, W6, V197, D175 SAS: 339, Size 17.75: L251, R180, N178, R10, W6, V197, D175 Epi#34 SAS: 430, Size 18.33: V238, W235, S236, G144, R143, S139, S142 SAS: 430, Size 18.33: V238, W235, S236, G144, R143, S142, S139 SAS: 420, Size 13.98: V238, W235, S236, G144, R143, S142, T141 SAS: 420, Size 13.98: V238, W235, S236, G144, R143, T141, S142 SAS: 352, Size 18.33: V238, W235, S236, G144, R143, S139, T141 Epi#37 SAS: 415, Size 23.06: T254, A188, L256, R180, N177 SAS: 374, Size 18.08: T254, A188, L256, R180, N179 SAS: 335, Size 19.96: T254, A188, L256, R180, N178 Epi#39 SAS: 425, Size 16.00: A166, E134, R164, P127, G125, L124 SAS: 421, Size 16.36: A131, E134, R164, P127, G125, L124 SAS: 400, Size 16.00: A166, E134, R164, P129, G125, L124 SAS: 396, Size 16.36: A131, E134, R164, P129, G125, L124 SAS: 359, Size 16.00: A166, E134, T132, P129, G125, L124 Epi#40 SAS: 358, Size 15.76: A166, G189, Y186, A188, T254 SAS: 352, Size 15.76: A166, G189, T254, A188, S158 SAS: 326, Size 11.62: A96, G59, T56, P54, S55 SAS: 322, Size 15.30: G98, G59, T56, P54, S55 SAS: 318, Size 17.81: A188, G189, Y186, A156, S182 Epi#42 SAS: 528, Size 16.22: L21, P14, S9, Q12, H17, R19, R269 Epi#44 SAS: 401, Size 15.10: L256, R180, Y186, S158, A188, T254 SAS: 393, Size 15.52: L256, R180, Y186, A188, G189, T254 SAS: 390, Size 18.46: L251, R180, Y186, S158, A188, T254 SAS: 382, Size 16.23: L251, R180, Y186, A188, G189, T254 SAS: 376, Size 22.23: V197, R180, Y186, S158, A188, T254 Epi#46 SAS: 559, Size 12.63: A15, R269, R19, P14, N18, G20 Epi#53 SAS: 298, Size 9.48: W235, S234, Q230, K231 SAS: 298, Size 18.05: W235, S234, Q239, K245 SAS: 289, Size 9.48: W235, P233, Q230, K231 SAS: 283, Size 9.61: W235, S234, Q239, K229 SAS: 255, Size 14.51: W235, S236, Q239, K245 ProteaseC: Epi#05 SAS: 445, Size 23.34: G189, A166, R164, P127, G125, S99 SAS: 445, Size 24.90: G189, A188, R164, P127, G125, S99 SAS: 433, Size 24.90: G157, A188, R164, P127, G125, S99 SAS: 427, Size 15.89: G189, A188, R164, P127, G125, S128 SAS: 427, Size 15.50: G189, A166, R164, P127, G125, S128 Epi#09 SAS: 463, Size 15.74: T254, G189, A166, R164, A188, S158 SAS: 425, Size 15.74: D191, G189, A166, R164, A188, T254 SAS: 384, Size 13.57: D191, G189, A166, R164, A188, S158 Epi#10 SAS: 445, Size 17.28: D175, N177, N179, S182, F183, G155, R180 SAS: 431, Size 13.75: D175, N212, N153, S182, F183, G155, R180 SAS: 403, Size 15.83: D175, N212, N153, S154, F183, G155, R180 SAS: 387, Size 16.14: D175, N178, N179, S182, F183, G155, R180 SAS: 373, Size 16.76: D175, N212, N153, A156, F183, G155, R180 Epi#12 SAS: 292, Size 13.45: P127, Y161, E134, P129 SAS: 287, Size 9.30: R44, Y89, E87, N42 SAS: 284, Size 9.35: R164, Y161, E134, P129 SAS: 282, Size 9.35: R164, Y165, E134, P129 SAS: 272, Size 16.85: P127, Y161, E134, N167 Epi#16 SAS: 547, Size 20.59: R164, P129, Y165, G189, S158, N255, D191, A166, N167 SAS: 543, Size 23.80: R164, P129, Y165, G189, S158, N255, D191, A166, N138 Epi#17 SAS: 267, Size 15.84: A188, S158, R164, S126 SAS: 231, Size 12.82: A156, S158, R164, S126 Epi#18 SAS: 449, Size 16.85: S182, R180, L256, A188, T254, L251 SAS: 426, Size 21.97: S126, R164, S158, A188, T254, L256 SAS: 407, Size 15.92: S182, R180, L251, G252, T254, L256 SAS: 407, Size 15.92: S182, R180, L256, G252, T254, L251 SAS: 391, Size 18.26: S182, R180, L256, G252, S250, L251 Epi#19 SAS: 293, Size 15.04: D175, W6, S9, Q12, R10 SAS: 291, Size 17.13: D191, N242, S236, Q239, R241 SAS: 273, Size 21.24: E110, T141, S236, Q239, R241 Epi#23 SAS: 463, Size 19.84: R143, N114, E110, S139, Q135, A131 SAS: 451, Size 15.68: R164, N167, E134, S139, Q135, A131 SAS: 443, Size 21.95: R44, N42, E87, S24, Q230, P233 SAS: 440, Size 22.70: R143, N115, E110, S139, Q135, A131 SAS: 431, Size 15.11: R44, N42, E87, S85, L73, P39 Epi#28 SAS: 402, Size 18.79: G59, Q57, E53, F49, G46, R44 SAS: 384, Size 20.81: A96, Q57, E53, F49, G46, R44 SAS: 376, Size 18.79: A47, Q57, E53, F49, G46, R44 Epi#31 SAS: 348, Size 21.63: L256, R180, N178, R10, W6, V197, D175 SAS: 342, Size 17.75: L251, R180, N178, R10, W6, V197, D175 Epi#33 SAS: 399, Size 18.88: Q107, Y102, P129, S126, R164 SAS: 355, Size 15.95: Q135, Y165, P129, S126, R164 Epi#34 SAS: 424, Size 18.37: V238, W235, S236, G144, R143, S139, S142 SAS: 424, Size 18.37: V238, W235, S236, G144, R143, S142, S139 SAS: 408, Size 14.02: V238, W235, S236, G144, R143, S142, T141 SAS: 408, Size 14.02: V238, W235, S236, G144, R143, T141, S142 SAS: 346, Size 18.37: V238, W235, S236, G144, R143, T141, S139 Epi#37 SAS: 405, Size 23.05: T254, A188, L256, R180, N177 SAS: 364, Size 18.08: T254, A188, L256, R180, N179 SAS: 347, Size 19.96: T254, A188, L256, R180, N178 Epi#40 SAS: 368, Size 15.74: A166, G189, T254, A188, S158 SAS: 362, Size 15.74: A166, G189, Y186, A188, T254 SAS: 326, Size 17.80: A188, G189, Y186, A156, S182 SAS: 326, Size 23.72: A166, G189, Y186, A156, S182 SAS: 326, Size 17.80: G189, A188, Y186, A156, S182 Epi#41 SAS: 232, Size 19.49: P204, Y208, L211, V197, S210 Epi#44 SAS: 445, Size 22.71: V238, R241, D191, Y186, S158, A188, T254 SAS: 429, Size 21.14: V238, R241, D191, Y186, A188, G189, T254 SAS: 410, Size 22.71: V238, R241, D191, Y186, S158, G189, T254 SAS: 404, Size 23.33: V238, R241, D191, Y257, S250, G252, T254 SAS: 382, Size 23.33: V238, R241, D191, Y257, S253, G252, T254 Epi#46 SAS: 567, Size 12.67: A15, R269, R19, P14, N18, G20 Epi#53 SAS: 305, Size 9.43: W235, S234, Q230, K231 SAS: 303, Size 9.53: W235, S234, Q239, K229 SAS: 276, Size 9.43: W235, P233, Q230, K231 SAS: 259, Size 9.43: W235, S234, Q230, K229 SAS: 233, Size 9.53: W235, S236, Q239, K229 ProteaseD: Epi#05 SAS: 453, Size 24.94: G189, A188, R164, P127, G125, S99 SAS: 449, Size 23.37: G189, A166, R164, P127, G125, S99 SAS: 442, Size 24.94: G157, A188, R164, P127, G125, S99 SAS: 439, Size 15.91: G189, A188, R164, P127, G125, S128 SAS: 435, Size 15.50: G189, A166, R164, P127, G125, S128 Epi#09 SAS: 448, Size 15.77: T254, G189, A166, R164, A188, S158 Epi#10 SAS: 460, Size 17.32: D175, N177, N179, S182, F183, G155, R180 SAS: 428, Size 13.76: D175, N212, N153, S182, F183, G155, R180 SAS: 403, Size 15.83: D175, N212, N153, S154, F183, G155, R180 SAS: 391, Size 16.15: D175, N178, N179, S182, F183, G155, R180 SAS: 372, Size 16.77: D175, N212, N153, A156, F183, G155, R180 Epi#12 SAS: 302, Size 13.47: P127, Y161, E134, P129 SAS: 290, Size 9.39: R164, Y161, E134, P129 SAS: 282, Size 18.68: P127, Y161, E134, N138 SAS: 280, Size 16.87: P127, Y161, E134, N167 SAS: 270, Size 13.10: R164, Y161, E134, N138 Epi#17 SAS: 286, Size 15.87: A188, S158, R164, S126 SAS: 250, Size 12.76: A156, S158, R164, S126 Epi#18 SAS: 446, Size 16.31: S250, K245, S259, L256, A188, T254, L251 SAS: 406, Size 14.13: S250, K245, S259, L256, G252, T254, L251 SAS: 406, Size 14.13: S250, K245, S259, L251, G252, T254, L256 SAS: 406, Size 14.13: S259, K245, S250, L251, G252, T254, L256 SAS: 388, Size 14.13: S250, K245, S259, L256, G252, T249, L251 Epi#19 SAS: 319, Size 15.07: D175, W6, S9, Q12, R10 SAS: 276, Size 21.28: E110, T141, S236, Q239, R241 Epi#23 SAS: 497, Size 19.86: R143, N114, E110, S139, Q135, A131 SAS: 487, Size 15.77: R164, N167, E134, S139, Q135, A131 SAS: 478, Size 13.78: R164, N167, E134, S130, Q135, A131 SAS: 477, Size 18.16: R143, N138, E134, S139, Q135, A131 SAS: 472, Size 22.70: R143, N115, E110, S139, Q135, A131 Epi#28 SAS: 554, Size 22.17: A101, Q107, I102, E134, Q135, S139, R143 SAS: 532, Size 19.36: 1102, Q107, W111, E110, Q135, S139, R143 SAS: 527, Size 22.79: V50, Q107, I102, E134, Q135, S139, R143 SAS: 509, Size 24.76: 1102, Q107, F49, E53, Q57, G46, R44 SAS: 508, Size 22.17: A101, Q107, W111, E110, Q135, S139, R143 Epi#31 SAS: 355, Size 21.56: L256, R180, N178, R10, W6, V197, D175 SAS: 352, Size 17.71: L251, R180, N178, R10, W6, V197, D175 Epi#34 SAS: 457, Size 18.37: V238, W235, S236, G144, R143, S139, S142 SAS: 457, Size 18.37: V238, W235, S236, G144, R143, S142, S139 SAS: 447, Size 14.02: V238, W235, S236, G144, R143, S142, T141 SAS: 447, Size 14.02: V238, W235, S236, G144, R143, T141, S142 SAS: 374, Size 18.37: V238, W235, S236, G144, R143, T141, S139 Epi#37 SAS: 397, Size 23.08: T254, A188, L256, R180, N177 SAS: 361, Size 18.08: T254, A188, L256, R180, N179 SAS: 328, Size 19.98: T254, A188, L256, R180, N178 Epi#39 SAS: 425, Size 16.36: A131, E134, R164, P127, G125, L124 SAS: 423, Size 16.02: A166, E134, R164, P127, G125, L124 SAS: 399, Size 16.36: A131, E134, R164, P129, G125, L124 SAS: 397, Size 16.02: A166, E134, R164, P129, G125, L124 SAS: 379, Size 16.36: A131, E134, T132, P129, G125, L124 Epi#40 SAS: 354, Size 15.77: A166, G189, T254, A188, S158 SAS: 351, Size 15.77: A166, G189, Y186, A188, T254 SAS: 334, Size 17.81: G189, A188, Y186, A156, S182 SAS: 334, Size 17.81: A188, G189, Y186, A156, S182 SAS: 330, Size 14.42: A166, G189, Y186, A188, S158 Epi#41 SAS: 217, Size 19.46: P204, Y208, L211, V197, S210 Epi#44 SAS: 407, Size 15.10: L256, R180, Y186, S158, A188, T254 SAS: 404, Size 18.45: L251, R180, Y186, S158, A188, T254 SAS: 387, Size 15.52: L256, R180, Y186, A188, G189, T254 SAS: 384, Size 16.23: L251, R180, Y186, A188, G189, T254 SAS: 373, Size 22.26: V197, R180, Y186, S158, A188, T254 Epi#46 SAS: 545, Size 12.69: A15, R269, R19, P14, N18, G20 Epi#53 SAS: 306, Size 18.06: W235, S234, Q239, K245 SAS: 277, Size 9.52: W235, S234, Q239, K229 SAS: 276, Size 9.46: W235, S234, Q230, K231 SAS: 268, Size 9.46: W235, P233, Q230, K231 SAS: 258, Size 14.50: W235, S236, Q239, K245 ProteaseE: Epi#05 SAS: 461, Size 15.49: G189, A166, R164, P127, G125, S128 SAS: 459, Size 15.90: G189, A188, R164, P127, G125, S128 SAS: 435, Size 15.49: G189, A166, R164, P127, G125, S126 SAS: 433, Size 15.49: G189, A166, R164, P129, G125, S128 SAS: 433, Size 15.86: G189, A188, R164, P127, G125, S126 Epi#06 SAS: 518, Size 14.10: G189, A188, D157, S158, R164, P127 SAS: 490, Size 15.98: G189, A188, D157, S158, R164, P129 SAS: 460, Size 14.60: G155, A156, D157, S158, R164, P127 SAS: 432, Size 17.71: G155, A156, D157, S158, R164, P129 Epi#09 SAS: 482, Size 15.78: T254, G189, A166, R164, A188, S158 SAS: 311, Size 15.91: T22, G20, L21, R19, A15, S9 Epi#10 SAS: 455, Size 17.26: D175, N177, N179, S182, F183, G155, R180 SAS: 406, Size 13.76: D175, N212, N153, S182, F183, G155, R180 SAS: 383, Size 16.16: D175, N178, N179, S182, F183, G155, R180 SAS: 381, Size 15.82: D175, N212, N153, S154, F183, G155, R180 SAS: 347, Size 16.78: D175, N212, N153, A156, F183, G155, R180 Epi#12 SAS: 310, Size 13.48: P127, Y161, E134, P129 SAS: 306, Size 9.40: R164, Y161, E134, P129 SAS: 297, Size 9.40: R164, Y165, E134, P129 SAS: 285, Size 16.90: P127, Y161, E134, N167 SAS: 281, Size 18.68: P127, Y161, E134, N138 Epi#16 SAS: 673, Size 19.67: R164, P127, Y161, G125, S126, S154, D157, A188, N255 SAS: 664, Size 20.60: R164, P129, Y165, G189, S158, S154, D157, A188, N255 SAS: 645, Size 20.60: R164, P129, Y161, G125, S126, S154, D157, A188, N255 SAS: 636, Size 14.89: R164, P127, Y161, G125, S126, S154, D157, A156, N153 SAS: 627, Size 17.25: R164, P129, Y165, G189, S158, S154, D157, A156, N153 Epi#17 SAS: 305, Size 15.86: A188, S158, R164, S126 SAS: 270, Size 12.73: A156, S158, R164, S126 Epi#18 SAS: 590, Size 17.32: S250, K246, S259, L256, A188, T254, L251 SAS: 551, Size 16.26: S259, K246, S250, L251, G252, T254, L256 SAS: 551, Size 16.26: S250, K246, S259, L251, G252, T254, L256 SAS: 551, Size 16.26: S250, K246, S259, L256, G252, T254, L251 SAS: 518, Size 16.26: S250, K246, S259, L251, G252, S253, L256 Epi#23 SAS: 471, Size 19.86: R143, N114, E110, S139, Q135, A131 SAS: 467, Size 13.75: R164, N167, E134, S130, Q135, A131 SAS: 467, Size 15.76: R164, N167, E134, S139, Q135, A131 SAS: 451, Size 22.69: R143, N115, E110, S139, Q135, A131 SAS: 446, Size 19.99: R143, N138, E134, S130, Q135, A131 Epi#28 SAS: 505, Size 19.43: 1102, Q107, W111, E110, Q135, S139, R143 SAS: 500, Size 22.22: A101, Q107, W111, E110, Q135, S139, R143 SAS: 499, Size 24.79: 1102, Q107, F49, E53, Q57, G46, R44 SAS: 494, Size 24.56: A101, Q107, F49, E53, Q57, G46, R44 SAS: 441, Size 24.79: 1102, Q107, E110, W111, F49, G46, R44 Epi#29 SAS: 216, Size 9.94: 143, R44, L41, E87 SAS: 209, Size 10.85: L73, N42, L41, E87 SAS: 200, Size 13.98: G46, R44, L41, E87 SAS: 199, Size 11.98: G45, R44, L41, E87 SAS: 197, Size 19.08: 177, N74, L41, E87 Epi#30 SAS: 318, Size 24.25: G20, L21, A15, H17, S85, L73, P39 SAS: 277, Size 24.25: G20, L21, A15, H17, S85, L41, P39 SAS: 258, Size 21.05: G20, L21, A15, H17, S85, L73, L41 Epi#31 SAS: 377, Size 21.62: L256, R180, N178, R10, W6, V197, D175 SAS: 370, Size 17.72: L251, R180, N178, R10, W6, V197, D175 Epi#33 SAS: 388, Size 15.92: Q135, Y165, P129, S126, R164 Epi#34 SAS: 420, Size 18.35: V238, W235, S236, G144, R143, S139, S142 SAS: 411, Size 13.98: V238, W235, S236, G144, R143, S142, T141 SAS: 341, Size 18.35: V238, W235, S236, G144, R143, S139, T141 Epi#37 SAS: 412, Size 23.05: T254, A188, L256, R180, N177 SAS: 378, Size 18.07: T254, A188, L256, R180, N179 SAS: 340, Size 20.00: T254, A188, L256, R180, N178 Epi#39 SAS: 445, Size 16.04: A166, E134, R164, P127, G125, L124 SAS: 432, Size 16.40: A131, E134, R164, P127, G125, L124 SAS: 417, Size 16.04: A166, E134, R164, P129, G125, L124 SAS: 404, Size 16.40: A131, E134, R164, P129, G125, L124 SAS: 376, Size 16.04: A166, E134, T132, P129, G125, L124 Epi#40 SAS: 374, Size 15.78: A166, G189, T254, A188, S158 SAS: 334, Size 15.78: A166, G189, Y186, A188, T254 SAS: 317, Size 11.62: A96, G59, T56, P54, S55 SAS: 312, Size 15.30: G98, G59, T56, P54, S55 SAS: 307, Size 15.49: G189, A166, Y165, P129, S128 Epi#41 SAS: 234, Size 19.50: P204, Y208, L211, V197, S210 SAS: 189, Size 19.50: P204, Y208, L211, V197, S215 Epi#42 SAS: 549, Size 16.42: L21, P14, S9, Q12, H17, R19, R269 Epi#44 SAS: 398, Size 15.10: L256, R180, Y186, S158, A188, T254 SAS: 391, Size 18.47: L251, R180, Y186, S158, A188, T254 SAS: 372, Size 15.51: L256, R180, Y186, A188, G189, T254 SAS: 371, Size 12.26: L256, R180, Y257, S250, G252, T254 SAS: 367, Size 15.51: L256, R180, Y186, S158, G189, T254 Epi#46 SAS: 575, Size 12.75: A15, R269, R19, P14, N18, G20 Epi#47 SAS: 491, Size 19.28: G45, E87, I43, R44, L41, N42, P39, S206 Epi#53 SAS: 202, Size 9.12: W235, P233, K231 SAS: 199, Size 9.12: W235, S234, K231 SAS: 182, Size 6.73: W235, P233, K229 SAS: 179, Size 7.76: W235, S234, K229 SAS: 131, Size 8.39: W235, S236, K229 Properase: Epi#05 SAS: 456, Size 15.94: G189, A188, R164, P127, G125, S128 SAS: 453, Size 15.52: G189, A166, R164, P127, G125, S128 SAS: 451, Size 15.94: G157, A188, R164, P127, G125, S128 SAS: 427, Size 15.94: G189, A188, R164, P129, G125, S128 SAS: 424, Size 15.52: G189, A166, R164, P129, G125, S128 Epi#09 SAS: 480, Size 15.73: T254, G189, A166, R164, A188, S158 SAS: 302, Size 15.88: T22, G20, L21, R19, A15, S9 Epi#10 SAS: 470, Size 17.27: D175, N177, N179, S182, F183, G155, R180 SAS: 446, Size 13.75: D175, N212, N153, S182, F183, G155, R180 SAS: 420, Size 15.84: D175, N212, N153, S154, F183, G155, R180 SAS: 396, Size 16.09: D175, N178, N179, S182, F183, G155, R180 SAS: 380, Size 16.78: D175, N212, N153, A156, F183, G155, R180 Epi#12 SAS: 296, Size 9.36: R164, Y161, E134, P129 SAS: 295, Size 13.45: P127, Y161, E134, P129 SAS: 291, Size 9.36: R164, Y165, E134, P129 SAS: 271, Size 14.70: R164, Y161, E134, N102 SAS: 270, Size 13.45: P127, Y161, E134, N102 Epi#17 SAS: 283, Size 15.87: A188, S158, R164, S126 SAS: 241, Size 12.73: A156, S158, R164, S126 Epi#18 SAS: 474, Size 16.26: S250, K245, S259, L256, A188, T254, L251 SAS: 435, Size 14.14: S250, K245, S259, L256, G252, T254, L251 SAS: 398, Size 14.14: S259, K245, S250, L251, G252, S253, L256 Epi#19 SAS: 260, Size 21.26: E110, T141, S236, Q239, R241 Epi#23 SAS: 491, Size 19.86: R143, N114, E110, S139, Q135, A131 SAS: 482, Size 15.76: R164, N167, E134, S139, Q135, A131 SAS: 465, Size 22.69: R143, N115, E110, S139, Q135, A131 SAS: 462, Size 18.17: R143, N138, E134, S139, Q135, A131 SAS: 439, Size 18.17: R143, N138, E110, S139, Q135, A131 Epi#28 SAS: 445, Size 22.79: V50, Q107, W111, E110, Q135, S139, R143 SAS: 426, Size 19.06: V50, Q107, F49, E53, Q57, G46, R44 SAS: 370, Size 19.06: V50, Q107, E110, W111, F49, G46, R44 Epi#31 SAS: 347, Size 21.62: L256, R180, N178, R10, W6, V197, D175 SAS: 339, Size 17.74: L251, R180, N178, R10, W6, V197, D175 Epi#33 SAS: 368, Size 15.95: Q135, Y165, P129, S126, R164 Epi#34 SAS: 445, Size 18.39: V238, W235, S236, G144, R143, S139, S142 SAS: 436, Size 14.07: V238, W235, S236, G144, R143, S142, T141 SAS: 358, Size 18.39: V238, W235, S236, G144, R143, T141, S139 Epi#37 SAS: 415, Size 23.03: T254, A188, L256, R180, N177 SAS: 374, Size 18.04: T254, A188, L256, R180, N179 SAS: 341, Size 19.93: T254, A188, L256, R180, N178 Epi#39 SAS: 323, Size 11.55: A15, E265, H17, R19, P14, G20, L21 SAS: 238, Size 12.13: A15, E265, H17, T22, P14, G20, L21 Epi#40 SAS: 370, Size 15.73: A166, G189, T254, A188, S158 SAS: 360, Size 15.73: A166, G189, Y186, A188, T254 SAS: 324, Size 17.80: A188, G189, Y186, A156, S182 SAS: 321, Size 23.71: A166, G189, Y186, A156, S182 Epi#41 SAS: 228, Size 19.53: P204, Y208, L211, V197, S210 Epi#42 SAS: 554, Size 16.31: L21, P14, S9, Q12, H17, R19, R269 Epi#44 SAS: 406, Size 15.06: L256, R180, Y186, S158, A188, T254 SAS: 398, Size 18.38: L251, R180, Y186, S158, A188, T254 SAS: 395, Size 12.22: L256, R180, Y257, S250, G252, T254 SAS: 392, Size 15.49: L256, R180, Y186, A188, G189, T254 SAS: 387, Size 12.22: L251, R180, Y257, S250, G252, T254 Epi#46 SAS: 581, Size 12.65: A15, R269, R19, P14, N18, G20 Epi#53 SAS: 297, Size 18.06: W235, S234, Q239, K245 SAS: 283, Size 9.54: W235, S234, Q239, K229 SAS: 250, Size 9.46: W235, S234, Q230, K231 SAS: 249, Size 14.49: W235, S236, Q239, K245 SAS: 247, Size 9.46: W235, P233, Q230, K231 Release: Epi#05 SAS: 461, Size 17.25: G158, A189, R165, P128, G126, S129 SAS: 439, Size 17.22: G158, A189, R165, P128, G126, S127 SAS: 436, Size 17.25: G158, A189, S159, P128, G126, S129 SAS: 420, Size 17.25: G158, A189, R165, P130, G126, S129 SAS: 414, Size 17.22: G158, A189, S159, P128, G126, S127 Epi#09 SAS: 510, Size 22.37: T22, G20, R19, A15, R270, A267, T250 SAS: 501, Size 22.37: L21, G20, R19, A15, R270, A267, T250 Epi#10 SAS: 458, Size 17.50: D176, N178, N180, S183, F184, G156, R181 SAS: 424, Size 13.68: D176, N213, N154, S183, F184, G156, R181 SAS: 407, Size 15.87: D176, N213, N154, S155, F184, G156, R181 SAS: 392, Size 16.18: D176, N179, N180, S183, F184, G156, R181 SAS: 362, Size 16.73: D176, N213, N154, A157, F184, G156, R181 Epi#12 SAS: 323, Size 9.38: R45, Y90, E88, N43 SAS: 312, Size 13.53: P128, Y162, E135, P130 SAS: 302, Size 9.46: R165, Y162, E135, P130 SAS: 296, Size 9.46: R165, Y166, E135, P130 SAS: 295, Size 13.19: T255, Y187, E190, S159 Epi#18 SAS: 431, Size 15.20: S251, K246, S260, L257, A189, T255, L252 SAS: 398, Size 14.35: S251, K246, S260, L252, G253, T255, L257 SAS: 378, Size 14.35: S251, K246, S260, L257, G253, T250, L252 Epi#19 SAS: 285, Size 21.53: E111, T142, S237, Q240, R242 SAS: 275, Size 12.58: D119, T142, S237, Q240, R242 Epi#23 SAS: 512, Size 22.29: R45, N43, E88, S24, Q231, P234 SAS: 476, Size 19.71: R144, N115, E111, S140, Q136, A132 SAS: 460, Size 13.83: R165, N168, E135, S131, Q136, A132 SAS: 455, Size 20.11: R144, N139, E135, S131, Q136, A132 SAS: 452, Size 15.83: R165, N168, E135, S140, Q136, A132 Epi#25 SAS: 293, Size 13.93: R45, K27, D119, E88 Epi#28 SAS: 502, Size 19.99: V103, Q108, W112, E111, Q136, S140, R144 SAS: 476, Size 21.74: V51, Q108, F50, E54, Q58, S37, R45 SAS: 472, Size 24.93: V103, Q108, F50, E54, Q58, G47, R45 SAS: 469, Size 23.18: V51, Q108, W112, E111, Q136, S140, R144 SAS: 439, Size 19.16: V51, Q108, F50, E54, Q58, G47, R45 Epi#31 SAS: 354, Size 21.73: L257, R181, N179, R10, W6, V198, D176 SAS: 348, Size 17.85: L252, R181, N179, R10, W6, V198, D176 Epi#33 SAS: 396, Size 22.75: Q201, Y204, P205, S37, R45 SAS: 379, Size 22.75: Q201, Y209, P205, S37, R45 SAS: 357, Size 18.39: H63, Y204, P205, S37, R45 Epi#34 SAS: 466, Size 13.97: V239, W236, S237, G145, R144, S143, T142 SAS: 463, Size 18.37: V239, W236, S237, G145, R144, S140, S143 SAS: 387, Size 18.37: V239, W236, S237, G145, R144, S140, T142 Epi#36 SAS: 206, Size 22.37: T250, A267, A15, G20, T22 Epi#37 SAS: 400, Size 22.59: T255, A189, L257, R181, N178 SAS: 359, Size 17.59: T255, A189, L257, R181, N180 SAS: 334, Size 19.35: T255, A189, L257, R181, N179 Epi#39 SAS: 464, Size 16.36: A167, E135, R165, P128, G126, L125 SAS: 444, Size 16.52: A132, E135, R165, P128, G126, L125 SAS: 441, Size 16.36: A167, E190, R165, P128, G126, L125 SAS: 441, Size 18.98: A189, E190, R165, P128, G126, L125 SAS: 423, Size 16.36: A167, E135, R165, P130, G126, L125 Epi#40 SAS: 324, Size 11.66: A97, G60, T57, P55, S56 SAS: 316, Size 17.09: G158, A189, Y187, A157, S183 SAS: 307, Size 14.92: G158, A157, Y187, A189, T255 SAS: 307, Size 15.34: G99, G60, T57, P55, S56 Epi#41 SAS: 222, Size 19.74: P205, Y209, L212, V198, S211 Epi#42 SAS: 544, Size 16.22: L21, P14, S9, Q12, H17, R19, R270 Epi#44 SAS: 421, Size 14.87: L257, R181, Y187, S159, A189, T255 SAS: 415, Size 18.81: L252, R181, Y187, S159, A189, T255 SAS: 389, Size 22.36: V198, R181, Y187, S159, A189, T255 SAS: 389, Size 21.81:144, R45, Y90, A48, V51, P52 SAS: 386, Size 19.16:144, R45, Y90, A48, V51, P55 Epi#46 SAS: 557, Size 14.54: A267, R270, R19, P14, N18, G20 SAS: 553, Size 12.63: A15, R270, R19, P14, N18, G20 SAS: 540, Size 13.10: A267, R270, R19, P14, N18, A15 SAS: 444, Size 14.54: A267, R270, R19, P14, G20, A15 Epi#47 SAS: 627, Size 16.22: A267, R270, A15, R19, L21, N18, P14, S9 SAS: 436, Size 15.11: A267, E266, A15, R19, L21, N18, P14, S9 Epi#51 SAS: 545, Size 21.66: L21, R19, H17, D75, S77, I78, S3, W6 SAS: 485, Size 21.66: L21, R19, H17, D75, Q2, I78, S3, W6 Epi#53 SAS: 328, Size 9.43: W236, S235, Q231, K232 SAS: 316, Size 9.43: W236, P234, Q231, K232 SAS: 301, Size 18.21: W236, S235, Q240, K246 SAS: 246, Size 14.68: W236, S237, Q240, K246

“SAS” is solvent accessible surface. “Size” is the total surface area of the epitope in A2.

Example 12

The object of this example is to provide evidence showing that subtilisins with an homology to BPN′ of as low as 44.8% reveal a similar epitope distribution as BPN′.

Alcalase, Protease B, Savinase, Esperase, and PD498 (which range from 44.8% to 69.5% in sequence identity to BPN′) were epitope mapped as described in the above example, and compared with epitope mapped BPN′ (FIG. 1).

The data in FIG. 1 show a significant overlap between the areas on the primary structure of the respective proteases. Overall, 6 regions were identified: 1-20, 35-65, 95-115, 130-145, 170-220, and 260-270.

Even better overlap between the epitope sequences can be found among proteins of higher sequence identity, such as within the Savinase-like subtilisins with more than 81% identity, preferably more than 85%, more preferably more than 90%, even more preferably more than 96% or most preferably more than 98% identity.

Example 13 Wash Performance

The following example provides results from a number of washing tests that were conducted under the conditions indicated

TABLE 9 Experimental conditions for evaluation of Subtilisin variants I44V. Detergent OMO Acao Detergent dose 2.5 g/l PH 10.5 Wash time 14 min. Temperature 25° C. Water hardness 9° dH Enzymes Subtilisin variant I44V Enzyme conc. 10 nM Test system 150 ml glass beakers with a stirring rod Textile/volume 5 textile pieces (Ø 2.5 cm) in 50 ml detergent Test material EMPA117 from Center for Test materials, Holland

TABLE 10 Experimental conditions for evaluation of Subtilisin variants Q12D. Detergent Persil Powder Detergent dose 4 g/l PH 10.5 Wash time 20 min. Temperature 30° C. Water hardness 18° dH Enzymes Subtilisin variant Q12D Enzyme conc. 10 nM Test system 150 ml glass beakers with a stirring rod Textile/volume 5 textile pieces (Ø 2.5 cm) in 50 ml detergent Test material EMPA116 from Center for Test materials, Holland

TABLE 11 Experimental conditions for evaluation of Subtilisin variants Q12D. Detergent Tide Detergent dose 1 g/l PH 10.5 Wash time 10 min. Temperature 25° C. Water hardness 6° dH Enzymes Subtilisin variant Q12D Enzyme conc. 10 nM Test system 150 ml glass beakers with a stirring rod Textile/volume 5 textile pieces (Ø 2.5 cm) in 50 ml detergent Test material EMPA117 from Center for Test materials, Holland

pH is adjusted to 10.5 which is within the normal range for a powder detergent.

Water hardness was adjusted by adding CaCl2 and MgCl2 (Ca2+:Mg2+=2:1) to deionized water (see also Surfactants in Consumer Products—Theory, Technology and Application, Springer Verlag 1986). pH of the detergent solution was adjusted to pH 10.5 by addition of HCl.

Measurement of reflectance (R) on the test material was done at 460 nm using a Macbeth ColorEye 7000 photometer. The measurements were done according to the manufacturers protocol.

The wash performance of the variants was evaluated by calculating a performance factor:


P=(RVariant−RBlank)/(RSavinase−RBlank)

P: Performance factor
RVariant: Reflectance of test material washed with variant
RSavinase: Reflectance of test material washed with Savinase®
RBlank: Reflectance of test material washed with no enzyme

The variants all have improved wash performance compared to Savinase®—i.e. P>1.

The variants can be divided into improvement classes designated with capital letters:

Class A: 1<P≦1.5 Class B: 1.5<P≦2 Class C: P>2

TABLE 12 Subtilisin variants and improvement classes. Improvement class Variants C I44V, Q12D

As it can be seen from Table 12 SAVINASE® variants of the invention exhibits an improvement in wash performance.

Claims

1-134. (canceled)

135. A protease variant having modified immunogenicity as compared to a parent protease, obtainable by a method comprising the steps of:

(a) obtaining antibody binding peptide sequences,
(b) using the sequences to localise epitope sequences on the 3-dimensional structure of the parent protein,
(c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence,
(d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein,
(e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and
(f) evaluating the immunogenicity of the protein variant using the parent protein as reference.

136. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position −6 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position −5 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position −4 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position −2 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 3a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 28a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 44a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 44b to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 139 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 148 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 149 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion;
Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion.

137. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position −1 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, H;
Position 1 to V, L, I, W, M, F, Y, S, T, R;
Position 2 to G, V, I, M, F, N, Q, Y, S, T, H;
Position 3 to W, M, F, N, Q, Y, S, D, E, R, H;
Position 4 to V, L, W, M, F, Y, R;
Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H;
Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H;
Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H;
Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R;
Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E;
Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H;
Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H;
Position 17 to G, A, V, I, W, P, M, F, Y, H;
Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H;
Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H;
Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E;
Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H;
Position 22 to G, V, L, I, W, M, F, Y, S, T;
Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R;
Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H;
Position 27 to G, L, I, W, P, M, F, Y, T, H;
Position 38 to V, L, I, W, M, F, N, Q, Y, T, H;
Position 39 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H;
Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H;
Position 42 to G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H;
Position 43 to G, L, H;
Position 44 to G, V, L, I, W, P, M, F, Y, S, T;
Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 46 to G, A, L, I, W, P, M, F, Y, H;
Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 48 to A, L, I, P, M, F, N, Y, D, H;
Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H;
Position 51 to V, L, I, W, M, F, N, Y, R;
Position 52 to V, L, I, W, M, F, Y, S, T, R;
Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H;
Position 54 to V, L, I, W, M, F, S, R;
Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H;
Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H;
Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 58 to L, W, M, F, N, Y, R;
Position 59 to A, V, L, I, C, T, H;
Position 61 to V, L, I, W, M, F, Y;
Position 62 to G, A, L, W, M, F, N, Y, R;
Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 75 to L;
Position 79 to I;
Position 80 to G;
Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H;
Position 89 to G, V, L, I, W, P, F, N, Y, T, E;
Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H;
Position 98 to A;
Position 99 to V, L, I, W, M, F, Q, Y, H;
Position 100 to G, V, L, I, W, M, F, Y, R, H;
Position 101 to V, I, W, M, F, N, Q, Y, H;
Position 102 to V, L, I, W, M, F, Y, R, H, G;
Position 108 to I;
Position 109 to N,
Position 112 to E;
Position 113 to W;
Position 115 to I;
Position 117 to N;
Position 118 to N;
Position 126 to L;
Position 127 to G, A, V, I, W, M, F, Y, R, H, L;
Position 128 to I, W;
Position 129 to W;
Position 130 to W, F, Y, R;
Position 131 to W, Y, R;
Position 132 to L, W, M, F, Y, S, H;
Position 133 to A, L, I, W, M, F, Y, R;
Position 134 to L, I, W, F, N, Q, Y, R, H;
Position 136 to G, A, W, P, N, Y, S, T, D, E, H;
Position 137 to G, A, V, I, W, P, M, N, Y, H;
Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H;
Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H;
Position 143 to V, L, I, P, M, F, N, Y, R;
Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H;
Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H;
Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H;
Position 155 to V, L, I, W, M, F, Y, R;
Position 156 to V, I, W, F, R;
Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H;
Position 158 to V, L, I, W, M, F, Y;
Position 159 to A, W, M, Y, T, R, H;
Position 160 to W, M, F, Y, R, H;
Position 161 to I, W, M, F, Y, H;
Position 167 to R, K;
Position 171 to D;
Position 172 to G, A, V, L, I, S, T, H;
Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H;
Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H;
Position 182 to A, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, H;
Position 183 to G, A, V, L, W, C, M, F, N, Q, Y, S, T, E, R, H;
Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H;
Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H;
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H;
Position 188 to G, A, V, L, W, F, S, R, K;
Position 189 to W, F;
Position 191 to A, V, L, I, W, M, F, Y, T, R, H;
Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H;
Position 194 to W, N, Q, Y, D, H;
Position 195 to W, P, Y;
Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 203 to V, F, Y, R, H;
Position 204 to I, W, M, Y, H;
Position 206 to F;
Position 209 to Y, R;
Position 210 to W, F, Y;
Position 211 to L, W, M, F, Y, H;
Position 212 to V, L, I, W, M, F, Y, T, R, H;
Position 214 to W, Y, R;
Position 215 to A, L, I, W, M, F, Y;
Position 216 to A, L, I, W, M, F, Y, R;
Position 217 to W, R;
Position 218 to G, A, L, W, P, M, F, Y, R, H;
Position 221 to S;
Position 236 to S;
Position 240 to N;
Position 241 to W;
Position 243 to N;
Position 245 to Q;
Position 247 to G, V, I, W, P, F, Y, S, T, R;
Position 248 to W, P, F, Y, E, R, H;
Position 249 to L, W, P, F, S, D, E, H;
Position 251 to G, L, I, W, P, M, F, Y, H;
Position 252 to G, A, W, P, N, Q, Y, T, E, R, H;
Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H;
Position 255 to G, L, W, M, F, N, Y, T, D, H;
Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D, H;
Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H;
Position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H;
Position 259 to A, V, I, W, M, F, N, Q, Y, S, T, E, R;
Position 260 to L, I, W, M, F, Y, T, H;
Position 261 to L, N, S, H;
Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H;
Position 263 to G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K;
Position 265 to V, L, I, W, M, F, Y;
Position 269 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, R, H;
Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H;
Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H;
Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D.

138. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position −1 to Deletion;
Position 9 to Insertion, deletion;
Position 10 to Insertion, deletion;
Position 12 to Insertion, deletion;
Position 14 to Insertion, deletion;
Position 15 to Insertion, deletion;
Position 17 to Insertion, deletion;
Position 18 to Insertion, deletion;
Position 19 to Insertion, deletion;
Position 20 to Insertion, deletion;
Position 21 to Insertion, deletion;
Position 22 to Insertion, deletion;
Position 24 to Insertion, deletion;
Position 25 to Insertion, deletion;
Position 46 to Insertion, deletion;
Position 47 to Insertion, deletion;
Position 48 to Insertion, deletion;
Position 49 to Insertion, deletion;
Position 50 to Insertion, deletion;
Position 51 to Insertion, deletion;
Position 52 to Insertion, deletion;
Position 53 to Insertion, deletion;
Position 54 to Insertion, deletion;
Position 55 to Insertion, deletion;
Position 58 to Insertion, deletion;
Position 59 to Insertion, deletion;
Position 61 to Insertion, deletion;
Position 64 to Insertion, deletion;
Position 78 to Insertion:
Position 80 to Insertion;
Position 91 to Insertion, deletion;
Position 98 to Deletion;
Position 99 to Deletion;
Position 102 to Deletion;
Position 105 to Insertion;
Position 108 to Insertion;
Position 109 to Insertion;
Position 112 to Insertion;
Position 113 to Insertion;
Position 115 to Insertion;
Position 116 to Insertion;
Position 117 to Insertion;
Position 118 to Insertion;
Position 131 to Deletion;
Position 134 to Insertion, deletion;
Position 136 to Insertion, deletion;
Position 137 to Insertion, deletion;
Position 140 to Insertion, deletion;
Position 141 to Insertion, deletion;
Position 143 to Insertion, deletion;
Position 144 to Insertion, deletion;
Position 145 to Insertion, deletion;
Position 146 to Insertion, deletion;
Position 171 to Deletion;
Position 172 to Deletion;
Position 173 to Deletion;
Position 181 to Deletion;
Position 182 to Deletion;
Position 183 to Deletion;
Position 184 to Deletion;
Position 185 to Deletion;
Position 186 to Deletion;
Position 188 to Deletion;
Position 189 to Deletion;
Position 191 to Deletion;
Position 192 to Deletion;
Position 195 to Deletion;
Position 196 to Insertion, deletion;
Position 221 to Insertion;
Position 236 to Insertion;
Position 237 to Insertion;
Position 238 to Insertion;
Position 239 to Insertion;
Position 240 to Insertion;
Position 241 to Insertion;
Position 242 to Insertion;
Position 243 to Insertion;
Position 244 to Insertion;
Position 245 to Insertion;
Position 247 to Insertion, deletion;
Position 248 to Insertion, deletion;
Position 249 to Insertion, deletion;
Position 251 to Insertion, deletion;
Position 252 to Insertion, deletion;
Position 254 to Insertion, deletion;
Position 255 to Insertion, deletion;
Position 256 to Insertion, deletion;
Position 257 to Insertion, deletion;
Position 258 to Insertion, deletion;
Position 259 to Insertion, deletion;
Position 260 to Insertion, deletion;
Position 261 to Insertion, deletion;
Position 262 to Insertion, deletion;
Position 263 to Insertion, deletion;
Position 265 to Insertion, deletion;
Position 269 to Insertion, deletion;
Position 271 to Insertion, deletion;
Position 272 to Insertion, deletion;
Position 275 to Insertion, deletion.

139. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position 7 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 8 to G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 13 to G, L, I, W, P, M, F, N, Q, Y, S, D, E, H;
Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H;
Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H;
Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H;
Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 29 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 33 to V, L, I, W, C, M, F, N, Q, Y, R, H;
Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 36 to V, L, I, W, P, M, F, N, Y, S, T, R, H;
Position 37 to L, I, W, M, F, N, Q, Y, S, R, H;
Position 41 to G, V, L, I, W, M, F, N, Q, Y, S, T, R, H;
Position 60 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H;
Position 63 to G, A, V, L, I, W, M, F, Y, T, R, H;
Position 73 to A;
Position 74 to A;
Position 81 to V;
Position 82 to L;
Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H;
Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H;
Position 92 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 93 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 94 to G, V, L, I, W, P, M, F, N, Y, T, D, E, K, H;
Position 96 to L, W, F, Y, R, K;
Position 97 to V, L, W, C, M, F, Y, H;
Position 111 to I;
Position 114 to A;
Position 119 to M;
Position 124 to M;
Position 135 to G, L, P, C, N, Q, T, R, H;
Position 138 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 151 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 162 to I, W, F, Y, R;
Position 163 to V, W, M, F, H;
Position 168 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y;
Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 176 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 179 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 187 to A, V, L, I, W, M, F, Y, R;
Position 190 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, R, K, H;
Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H;
Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H;
Position 198 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 205 to W, F, Y, R, K;
Position 208 to A, V, L, I, W, C, M, F, Y, T, R, K, H;
Position 219 to G, A, V, L, I, W, F, Y, R, H;
Position 222 to M;
Position 232 to A;
Position 233 to L;
Position 234 to I;
Position 250 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H;
Position 267 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H;
Position 268 to G, V, L, I, W, C, M, N, Q, Y, S, T, D, E, R, K, H;
Position 270 to G, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H;
Position 274 to W, P, M, F, N, Q, Y, T, D, E, R, H.

140. The protease variant of claim 135, wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position 13 to Insertion, deletion;
Position 16 to Insertion, deletion;
Position 23 to Insertion, deletion;
Position 26 to Insertion, deletion;
Position 28 to Insertion, deletion;
Position 29 to Insertion, deletion;
Position 35 to Deletion;
Position 60 to Insertion, deletion;
Position 63 to Insertion;
Position 81 to Insertion;
Position 82 to Insertion;
Position 92 to Insertion, deletion;
Position 93 to Insertion, deletion;
Position 94 to Insertion, deletion;
Position 96 to Deletion,
Position 106 to Insertion,
Position 111 to Insertion,
Position 114 to Insertion,
Position 119 to Insertion,
Position 124 to Insertion,
Position 138 to Insertion, deletion;
Position 142 to Insertion, deletion;
Position 147 to Insertion, deletion;
Position 151 to Insertion, deletion;
Position 174 to Insertion, deletion;
Position 176 to Insertion, deletion;
Position 179 to Insertion, deletion;
Position 187 to Deletion;
Position 190 to Deletion;
Position 193 to Deletion;
Position 197 to Insertion, deletion;
Position 198 to Insertion, deletion;
Position 232 to Insertion,
Position 233 to Insertion,
Position 234 to Insertion,
Position 246 to Insertion,
Position 250 to Insertion, deletion;
Position 267 to Insertion, deletion;
Position 268 to Insertion, deletion;
Position 270 to Insertion, deletion;
Position 273 to Insertion, deletion.

141. The protease variant of claim 135, wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position 2 to G, V, I, M, F, N, Q, Y, S, T, H,
Position 3 to W, M, F, N, Q, Y, S, D, E, R, H,
Position 4 to V, L, W, M, F, Y, R,
Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H,
Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion,
Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion,
Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,
Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion, deletion,
Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion,
Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,
Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion,
Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, insertion, deletion,
Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 27 to G, L, I, W, P, M, F, Y, T, H,
Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,
Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 42 to G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H,
Position 43 to G, L, H,
Position 44 to G, V, L, I, W, P, M, F, Y, S, T,
Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 46 to G, A, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 48 to A, L, I, P, M, F, N, Y, D, H, insertion, deletion,
Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 54 to V, L, I, W, M, F, S, R, deletion, insertion,
Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion,
Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 58 to L, W, M, F, N, Y, R, insertion, deletion,
Position 59 to A, V, L, I, C, T, H, insertion, deletion,
Position 61 to V, L, I, W, M, F, Y, insertion, deletion,
Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 75 to L,
Position 78 to insertion,
Position 79 to I,
Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,
Position 89 to G, V, L, I, W, P, F, N, Y, T, E,
Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion,
Position 98 to A, deletion,
Position 100 to G, V, L, I, W, M, F, Y, R, H,
Position 101 to V, I, W, M, F, N, Q, Y, H,
Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion,
Position 109 to N, insertion,
Position 112 to E, insertion,
Position 113 to W, insertion,
Position 116 to insertion,
Position 117 to N, insertion,
Position 126 to L,
Position 127 to G, A, V, I, W, M, F, Y, R, H, L,
Position 128 to I, W,
Position 129 to W,
Position 130 to W, F, Y, R,
Position 131 to W, Y, R, deletion,
Position 132 to L, W, M, F, Y, S, H,
Position 133 to A, L, I, W, M, F, Y, R,
Position 134 to L, I, W, F, N, Q, Y, R, H, insertion, deletion,
Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H, insertion, deletion,
Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 143 to V, L, I, P, M, F, N, Y, R, insertion, deletion,
Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,
Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 155 to V, L, I, W, M, F, Y, R,
Position 156 to V, I, W, F, R,
Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H,
Position 158 to V, L, I, W, M, F, Y,
Position 160 to W, M, F, Y, R, H,
Position 161 to I, W, M, F, Y, H,
Position 167 to R, K,
Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
Position 171 to D, deletion,
Position 172 to G, A, V, L, I, S, T, H, deletion,
Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,
Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion,
Position 183 to G, A, V, L, W, C, M, F, N, Q, Y, S, T, E, R, H, deletion,
Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to G, A, V, L, W, F, S, R, K, deletion,
Position 189 to W, F, deletion,
Position 191 to A, V, L, I, W, M, F, Y, T, R, H, deletion,
Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 194 to W, N, Q, Y, D, H,
Position 195 to W, P, Y, deletion,
Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 203 to V, F, Y, R, H,
Position 206 to F,
Position 209 to Y, R,
Position 210 to W, F, Y,
Position 212 to V, L, I, W, M, F, Y, T, R, H,
Position 214 to W, Y, R,
Position 216 to A, L, I, W, M, F, Y, R,
Position 217 to W, R,
Position 218 to G, A, L, W, P, M, F, Y, R, H,
Position 221 to S, insertion,
Position 236 to S, insertion,
Position 237 to insertion,
Position 239 to insertion,
Position 240 to N, insertion,
Position 241 to W, insertion,
Position 242 to insertion,
Position 244 to insertion,
Position 245 to Q, insertion,
Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion,
Position 248 to W, P, F, Y, E, R, H, insertion, deletion,
Position 251 to G, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,
Position 255 to G, L, W, M, F, N, Y, T, D, H, insertion, deletion,
Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D, H, insertion, deletion,
Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion,
Position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H, insertion, deletion,
Position 259 to A, V, I, W, M, F, N, Q, Y, S, T, E, R, insertion, deletion,
Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion,
Position 261 to L, N, S, H, insertion, deletion,
Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 263 to G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K, insertion, deletion,
Position 265 to V, L, I, W, M, F, Y, insertion, deletion,
Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion,
Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion.

142. The protease variant of claim 141, wherein the savinase-like subtilisin comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H,
Position 9 to G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion,
Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion,
Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,
Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion, deletion,
Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion,
Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,
Position 20 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,
Position 43 to G, L, H,
Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 54 to V, L, I, W, M, F, S, R, deletion, insertion,
Position 59 to A, V, L, I, C, T, H, insertion, deletion,
Position 89 to G, V, L, I, W, P, F, N, Y, T, E,
Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion,
Position 101 to V, I, W, M, F, N, Q, Y, H,
Position 109 to N, insertion,
Position 112 to E, insertion,
Position 113 to W, insertion,
Position 127 to G, A, V, I, W, M, F, Y, R, H, L,
Position 128 to I, W,
Position 129 to W,
Position 130 to W, F, Y, R,
Position 131 to W, Y, R, deletion,
Position 133 to A, L, I, W, M, F, Y, R,
Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 140 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H, insertion, deletion,
Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 143 to V, L, I, P, M, F, N, Y, R, insertion, deletion,
Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,
Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 155 to V, L, I, W, M, F, Y, R,
Position 157 to G, A, V, L, I, W, M, F, Y, T, R, H,
Position 158 to V, L, I, W, M, F, Y,
Position 160 to W, M, F, Y, R, H,
Position 161 to I, W, M, F, Y, H,
Position 167 to R, K,
Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
Position 171 to D, deletion,
Position 172 to G, A, V, L, I, S, T, H, deletion,
Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,
Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion,
Position 184 to A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to G, A, V, L, W, F, S, R, K, deletion,
Position 189 to W, F, deletion,
Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 194 to W, N, Q, Y, D, H,
Position 195 to W, P, Y, deletion,
Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 203 to V, F, Y, R, H,
Position 210 to W, F, Y,
Position 218 to G, A, L, W, P, M, F, Y, R, H,
Position 236 to S, insertion,
Position 237 to insertion,
Position 239 to insertion,
Position 240 to N, insertion,
Position 241 to W, insertion,
Position 242 to insertion,
Position 244 to insertion,
Position 245 to Q, insertion,
Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion,
Position 251 to G, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 255 to G, L, W, M, F, N, Y, T, D, H, insertion, deletion,
Position 256 to G, A, V, L, I, W, M, F, Q, Y, S, T, D, H, insertion, deletion,
Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion,
Position 258 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H, insertion, deletion,
Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion,
Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 265 to V, L, I, W, M, F, Y, insertion, deletion,
Position 271 to A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion,
Position 272 to G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion.

143. The savinase-like subtilisin of claim 141, wherein the subtilisin has at least 81% homology to SEQ ID NO: 24.

144. The savinase-like subtilisin of claim 141, wherein the subtilisin has any of the amino acid sequence of SEQ ID NO: 24, 26, 27, 28, 29, 30, 31, 32, 34, 35.

145. The protein variant of claim 135, wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:

Position 8 to G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion,
Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, deletion,
Position 38 to V, L, I, W, M, F, N, Q, Y, T, H,
Position 39 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
Position 41 to G, V, L, I, W, M, F, N, Q, Y, S, T, R, H,
Position 60 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, insertion, deletion,
Position 73 to A,
Position 74 to A,
Position 80 to G, insertion,
Position 81 to V, insertion,
Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,
Position 90 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 93 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 108 to I, insertion,
Position 111 to I, insertion,
Position 124 to M, insertion,
Position 135 to G, L, P, C, N, Q, T, R, H,
Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 148 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 149 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 151 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 163 to V, W, M, F, H,
Position 168 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y,
Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 179 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 190 to G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, R, K, H, deletion,
Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 208 to A, V, L, I, W, C, M, F, Y, T, R, K, H,
Position 213 to N, oN, E,
Position 215 to A, L, I, W, M, F, Y,
Position 232 to A, insertion,
Position 233 to L, insertion,
Position 234 to I, insertion,
Position 246 to insertion,
Position 250 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 267 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 268 to G, V, L, I, W, C, M, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 269 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, R, H, insertion, deletion,
Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion.

146. The savinase-like subtilisin of claim 145, wherein the subtilisin has at least 81% homology to SEQ ID NO: 24.

147. The savinase-like subtilisin of claim 146, wherein the subtilisin has any of the amino acid sequence of SEQ ID NO: 24, 26, 27, 28, 29, 30, 31, 32, 34, 35.

148. The protease variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 25 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 25: with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 27 to G, L, I, W, P, M, F, Y, T, H,
Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion,
Position 129 to W,
Position 133 to A, L, I, W, M, F, Y, R,
Position 172 to G, A, V, L, I, S, T, H, deletion,
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 194 to W, N, Q, Y, D, H,
Position 195 to W, P, Y, deletion,
Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 242 to insertion,
Position 249 to L, W, P, F, S, D, E, H, insertion, deletion,
Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,
Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion,
Position 260 to L, I, W, M, F, Y, T, H, insertion, deletion,
Position 265 to V, L, I, W, M, F, Y, insertion, deletion,

149. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 10 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10: with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

Position 4 to V, L, W, M, F, Y, R,
Position 38 to V, L, I, W, M, F, N, Q, Y, T, H,
Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 43 to G, L, H,
Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 54 to V, L, I, W, M, F, S, R, deletion, insertion,
Position 96 to L, W, F, Y, R, K, deletion,
Position 99 to V, L, I, W, M, F, Q, Y, H, deletion,
Position 113 to W, insertion,
Position 131 to W, Y, R, deletion,
Position 133 to A, L, I, W, M, F, Y, R,
Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
Position 173 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,
Position 181 to G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion,
Position 185 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to G, A, V, L, W, F, S, R, K, deletion,
Position 194 to W, N, Q, Y, D, H,
Position 203 to V, F, Y, R, H,
Position 210 to W, F, Y,
Position 211 to L, W, M, F, Y, H,
Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion,
Position 261 to L, N, S, H, insertion, deletion,
Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 265 to V, L, I, W, M, F, Y, insertion, deletion.

150. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 11 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 11: with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

Position 38 to V, L, I, W, M, F, N, Q, Y, T, H,
Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 45 to G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 47 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 49 to G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 50 to G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion, insertion,
Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H,
Position 58 to L, W, M, F, N, Y, R, insertion, deletion,
Position 96 to L, W, F, Y, R, K, deletion,
Position 97 to V, L, W, C, M, F, Y, H,
Position 98 to A, deletion,
Position 105 to insertion,
Position 109 to N, insertion,
Position 113 to W, insertion,
Position 115 to I, insertion,
Position 133 to A, L, I, W, M, F, Y, R,
Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 158 to V, L, I, W, M, F, Y,
Position 159 to A, W, M, Y, T, R, H,
Position 172 to G, A, V, L, I, S, T, H, deletion,
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 189 to W, F, deletion,
Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 195 to W, P, Y, deletion,
Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 257 to G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion, deletion,
Position 261 to L, N, S, H, insertion, deletion,
Position 265 to V, L, I, W, M, F, Y, insertion, deletion,

151. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 33: with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

Position −6 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y, insertion, deletion,
Position −5 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y, insertion, deletion,
Position −4 to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, insertion, deletion,
Position −2 to A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position −1 to G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 1 to V, L, I, W, M, F, Y, S, T, R,
Position 2 to G, V, I, M, F, N, Q, Y, S, T, H,
Position 3a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 6 to G, V, L, I, W, P, M, N, Q, T, D, E, R, H,
Position 7 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 8 to G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 10 to G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion,
Position 12 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 13 to G, L, I, W, P, M, F, N, Q, Y, S, D, E, H, insertion, deletion,
Position 14 to V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,
Position 15 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion, deletion,
Position 16 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 17 to G, A, V, I, W, P, M, F, Y, H, insertion, deletion,
Position 18 to G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 19 to A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,
Position 21 to G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion,
Position 23 to G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion,
Position 24 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, insertion, deletion,
Position 25 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 27 to G, L, I, W, P, M, F, Y, T, H,
Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 28a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 29 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 33 to V, L, I, W, C, M, F, N, Q, Y, R, H,
Position 35 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, deletion,
Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,
Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 42 to G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H,
Position 43 to G, L, H,
Position 44 to G, V, L, I, W, P, M, F, Y, S, T,
Position 44a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 44b to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 46 to G, A, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 48 to A, L, I, P, M, F, N, Y, D, H, insertion, deletion,
Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 53 to A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion, insertion,
Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion,
Position 56 to G, V, L, I, W, M, F, N, Q, Y, S, T, H,
Position 57 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 58 to L, W, M, F, N, Y, R, insertion, deletion,
Position 61 to V, L, I, W, M, F, Y, insertion, deletion,
Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 75 to L,
Position 81 to insertion,
Position 86 to G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,
Position 88 to A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,
Position 89 to G, V, L, I, W, P, F, N, Y, T, E,
Position 91 to G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion, deletion,
Position 92 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 94 to G, V, L, I, W, P, M, F, N, Y, T, D, E, K, H, insertion, deletion,
Position 96 to L, W, F, Y, R, K, deletion,
Position 97 to V, L, W, C, M, F, Y, H,
Position 98 to deletion,
Position 101 to V, I, W, M, F, N, Q, Y, H,
Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion,
Position 108 to I, insertion,
Position 109 to N, insertion,
Position 111 to insertion,
Position 112 to E, insertion,
Position 113 to W, insertion,
Position 114 to insertion,
Position 115 to I, insertion,
Position 117 to N, insertion,
Position 118 to N, insertion,
Position 119 to M, insertion,
Position 127 to G, A, V, I, W, M, F, Y, R, H, L,
Position 133 to A, L, I, W, M, F, Y, R,
Position 134 to L, I, W, F, N, Q, Y, R, H, insertion, deletion,
Position 135 to G, L, P, C, N, Q, T, R, H,
Position 136 to G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 138 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 139 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 141 to G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 142 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 144 to L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 145 to G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,
Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 147 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 148 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 156 to V, I, W, F, R,
Position 158 to V, L, I, W, M, F, Y,
Position 160 to W, M, F, Y, R, H,
Position 161 to I, W, M, F, Y, H,
Position 162 to I, W, F, Y, R,
Position 163 to V, W, M, F, H,
Position 167 to R, K,
Position 169 to C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y,
Position 170 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 171 to D, deletion,
Position 174 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 176 to G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 182 to A, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, H, deletion,
Position 186 to G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to G, A, V, L, W, F, S, R, K, deletion,
Position 191 to A, V, L, I, W, M, F, Y, T, R, H, deletion,
Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 193 to G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 194 to W, N, Q, Y, D, H,
Position 195 to W, P, Y, deletion,
Position 196 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 197 to G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 198 to G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 203 to V, F, Y, R, H,
Position 205 to W, F, Y, R, K,
Position 215 to A, L, I, W, M, F, Y,
Position 216 to A, L, I, W, M, F, Y, R,
Position 217 to W, R,
Position 219 to G, A, V, L, I, W, F, Y, R, H,
Position 233 to insertion,
Position 234 to I, insertion,
Position 236 to insertion,
Position 237 to insertion,
Position 238 to insertion,
Position 239 to insertion,
Position 240 to insertion,
Position 243 to insertion,
Position 246 to insertion,
Position 247 to G, V, I, W, P, F, Y, S, T, R, insertion, deletion,
Position 249 to L, W, P, F, S, D, E, H, insertion, deletion,
Position 252 to G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,
Position 254 to G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,
Position 270 to G, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 273 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 274 to W, P, M, F, N, Q, Y, T, D, E, R, H,
Position 275 to G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion,
Position 276 to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,

152. The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 33: with the proviso that the amino acids of the parent enzyme are substituted to another amino acid.

Position 5 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 22 to G, V, L, I, W, M, F, Y, S, T, insertion, deletion,
Position 26 to G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 28 to G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 37 to L, I, W, M, F, N, Q, Y, S, R, H,
Position 40 to V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 44 to G, V, L, I, W, P, M, F, Y, S, T,
Position 51 to V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 52 to V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 55 to G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion, insertion,
Position 58 to L, W, M, F, N, Y, R, insertion, deletion,
Position 61 to V, L, I, W, M, F, Y, insertion, deletion,
Position 64 to G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion, deletion,
Position 87 to A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,
Position 97 to V, L, W, C, M, F, Y, H,
Position 98 to deletion,
Position 101 to V, I, W, M, F, N, Q, Y, H,
Position 102 to V, L, I, W, M, F, Y, R, H, G, deletion,
Position 109 to N, insertion,
Position 112 to E, insertion,
Position 118 to N, insertion,
Position 127 to G, A, V, I, W, M, F, Y, R, H, L,
Position 137 to G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 146 to G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 156 to V, I, W, F, R,
Position 158 to V, L, I, W, M, F, Y,
Position 161 to I, W, M, F, Y, H,
Position 188 to G, A, V, L, W, F, S, R, K, deletion,
Position 192 to G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 194 to W, N, Q, Y, D, H,
Position 195 to W, P, Y, deletion,
Position 203 to V, F, Y, R, H,
Position 216 to A, L, I, W, M, F, Y, R,
Position 236 to insertion,
Position 237 to insertion,
Position 262 to G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 264a to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y, insertion, deletion,

153. A subtilisin variant comprising one or more of the insertions, substitutions and/or deletions in any of the positions of claim 136.

154. The variant of claim 153, wherein the subtilisin has at least 80% homology to SEQ ID NO; 10.

155. A composition comprising the protein variant of claim 135.

156. A DNA construct comprising a DNA sequence encoding a protein variant of claim 135.

157. An expression vector comprising a DNA construct of claim 156.

158. A host cell which is capable of expressing a polypeptide and comprising a DNA construct of claim 156.

159. A host cell which is capable of expressing a polypeptide and which is transformed by an expression vector of claim 157.

160. The host cell of claim 159, which is a fungal cell, an insect cell, a mammalian cell, or a plant cell.

161. A method of producing a protein variant having reduced immunogenicity as compared to the parent protein, comprising:

(a) culturing the host of claim 159 in a suitable culture medium to obtain expression and secretion of the protein into the medium, followed by
(b) isolation of the protein from the culture medium.
Patent History
Publication number: 20110045572
Type: Application
Filed: Feb 4, 2010
Publication Date: Feb 24, 2011
Applicant: NOVOZYMES A/S (BAGSVAERD)
Inventors: Erwin Ludo Roggen (Lyngby), Steffen Ernst (Bronshoj), Allan Svendsen (Horsholm), Esben Peter Friis (Valby), Claus Von Der Osten (Lyngby)
Application Number: 12/699,979