Mammalian cytokine-like receptor 5
Novel receptor polypeptides, polynucleotides encoding the polypeptides, and related compositions. The polypeptides of the present invention can be used to down-regulate their natural ligands. The polynucleotides and subsequences thereof can be used as diagnostic probes to determine if chromosome 19 is mutated. The antibodies which bind to the polypeptides can be used to purify the receptors and to inhibit the binding of the ligands onto the receptors.
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[0001] Proliferation and differentiation of cells of multicellular organisms are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
[0002] Hormones and growth factors influence cellular metabolism by binding to receptors. Receptors may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble molecules, such as the transcription factors.
[0003] Of particular interest are receptors for cytokines, molecules that promote the proliferation and/or differentiation of cells. Examples of cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF), which stimulates development of neutrophils. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia or receiving chemotherapy for cancer. The demonstrated in vivo activities of these cytokines illustrates the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists.
SUMMARY OF THE INVENTION[0004] The present invention addresses this need by providing a novel mammalian cytokine-like receptor called mammalian Zcytor5, and related compositions and methods. Within one aspect, the present invention provides an isolated human polynucleotide encoding a ligand-binding human receptor polypeptide. The polypeptide comprises a sequence of amino acids containing (a) the amino acid residues of SEQ ID NO:17, residues 35 to 422 of SEQ ID NO:2; (b) allelic variants of (a); and (c) sequences that are at least 90%, 95% or 99% identical to (a) or (b). In an alternative embodiment, the polypeptide is comprised of amino acid residues 30 to and including amino acid residue 422 of SEQ ID NO:2.
[0005] The present invention also provides for a polynucleotide encoding another allelic variant of SEQ ID NO:2 which is a human polypeptide receptor and is defined by SEQ ID NO:4 in particular the polypeptide comprised of a sequence of amino acids containing (a)the amino acid residues of SEQ ID NO:18, residues 34 to 425 of SEQ ID NO:4; (b) allelic variants of (a); and (c) sequences that are at least 90%, 95% or 99% identical to (a) or (b). In an alternative embodiment, the polypeptide is comprised of amino acid residues 29 to and including amino acid residue 425 of SEQ ID NO:4.
[0006] Other polynucleotides of the present invention encode the amino acid sequence of SEQ ID NO:21 which is a soluble receptor of SEQ ID NO:17 that does not contain a C-terminus phosphatidylinositol signal sequence; the amino acid sequence of SEQ ID NO:20 is a Zcytor5 polypeptide of SEQ ID NO:2 having an alternative N-terminus cleavage site; SEQ ID NO:22 which has an alternative N-terminus cleavage site of the Zcytor5 polypeptide of SEQ ID NO:4; SEQ ID NO:23 which is an amino acid of SEQ ID NO:18 that does not contain a C-terminus phosphatidylinositol signal sequence and the amino acid sequences defined by SEQ ID NOs: 24-31 which are variants of the Zcytor5 polypeptide of SEQ ID NO:4.
[0007] Another embodiment of the present invention is a polynucleotide which encodes rat Zcytor5. In particular, a polynucleotide is claimed which encodes a rat polypeptide containing (a)the amino acid sequence of SEQ ID NO:19 residues 41 to 425 of SEQ ID NO:6; (b) allelic variants of (a); and (c) sequences that are at least 90%, 95% or 99% identical to (a) or (b).
[0008] Within a second aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a secretory peptide and a ligand-binding Zcyotor5 receptor polypeptide, containing an amino acid sequence as described above.
[0009] Within a third aspect of the invention there is provided a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a mammalian Zcytor5 receptor polypeptide encoded by the DNA segment.
[0010] Within a fourth aspect of the invention there is provided an isolated polypeptide. The polypeptide comprises a sequence of amino acids containing (a) the amino acid sequence of SEQ ID NO:17, residues 35 to 422 of SEQ ID NO:2; (b) allelic variants of (a); and (c) sequences that are at least 90%, 95% or 99% identical to (a) or (b). In an alternative embodiment, the polypeptide is comprised of amino acid residues 30 to and including amino acid residue 422 of SEQ ID NO:2.
[0011] The present invention also provides for another allelic variant of SEQ ID NO:2 which is a human polypeptide receptor and is defined by SEQ ID NO:4 in particular the polypeptide is comprised of a sequence of amino acids containing (a) the amino acid sequence of SEQ ID NO:18, residues 34 to 425 of SEQ ID NO:4; (b) allelic variants of (a); and (c) sequences that are at least 90%, 95% or 99% identical to (a) or (b). In an alternative embodiment, the polypeptide is comprised of residues 29 to 425 of SEQ ID NO:4.
[0012] Another embodiment of the present invention is a rat Zcytor5 polypeptide containing (a) the amino acid sequence of SEQ ID NO:19, residues 41 to 425 of SEQ ID NO:6; (b) allelic variants of (a); and (c) sequences that are at least 80% identical to (a) or (b).
[0013] Within a further aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond. The first portion of the chimeric polypeptide consists essentially of a Zcytor5 receptor polypeptide as described above. The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
[0014] The invention also provides a method for detecting a ligand within a test sample, comprising contacting a test sample with a Zcytor5 polypeptide as disclosed above, and detecting binding of the polypeptide to ligand in the sample. The polypeptide can be membrane bound within a cultured cell, wherein the detecting step comprises measuring a biological response in the cultured cell. Within another embodiment, the polypeptide is immobilized on a solid support.
[0015] Within an additional aspect of the invention there is provided an antibody that specifically binds to a polypeptide as disclosed above and an anti-idiotypic antibody of an antibody which specifically binds to a Zcytor5 antibody, also a method for producing an antibody to Zcytor5.
[0016] An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zcytor5 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zcytor5 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Examples of said polypeptides are defined by the amino acid sequences of SEQ ID NOs: 32-37.
DETAILED DESCRIPTION OF THE INVENTION[0017] The teachings of all of the references cited in the present specification are incorporated in their entirety herein by reference.
[0018] The term “affinity tag” is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A [Nilsson et al, EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991)], glutathione S transferase [Smith and Johnson, Gene 67:31 (1988)], Glu-Glu affinity tag [Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)], substance P, FLAG™ peptide (Hopp et al., Biotechnology 6:1204-10 (1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107 (1991). DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).
[0019] The term “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 phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
[0020] The term “allelic variant” is used herein to denote 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 phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
[0021] The terms “amino-terminal” and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
[0022] The term “complement/anti-complement pair” denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M±1.
[0023] The term “complements of a polynucleotide molecule” is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
[0024] The term “contig” denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to “overlap” a given stretch of polynucleotide sequence either in their entirety or alone a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5′-ATGGCTTAGCTT -3′ are 5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.
[0025] The term “degenerate nucleotide sequence” denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
[0026] The term “expression vector” is used to denote 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 include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
[0027] The term “isolated”, when applied to a polynucleotide, denotes that the polynucleotide has been 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, but 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, for example, Dynan and Tijan, Nature 316:774-78 (1985).
[0028] An “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. “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.
[0029] The term “ortholog” (or “species homolog”) denotes a polypeptide or protein obtained from one species that has homology to an analogous polypeptide or protein from a different species.
[0030] The term “paralog” denotes a polypeptide or protein obtained from a given species that has homology to a distinct polypeptide or protein from that same species.
[0031] 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.
[0032] A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
[0033] 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.
[0034] A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
[0035] The term “receptor” denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
[0036] The term “secretory signal sequence” denotes 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.
[0037] The term “splice variant” is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
[0038] A “soluble receptor” is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
[0039] The present invention is based in part upon the discovery of a novel DNA sequence that encodes a protein having the structure of a cytokine receptor, including the conserved WSXWS motif. Analysis of the tissue distribution of the mRNA corresponding to this novel DNA showed that expression was present in highest amounts in placenta, thyroid, heart and skeletal muscle with lower levels in prostate and trachea.
[0040] Cytokine receptors subunits are characterized by a multi-domain structure comprising a ligand-binding domain and an effector domain that is typically involved in signal transduction. Multimeric cytokine receptors include homodimers (e.g., PDGF receptor ma and Ad &bgr;&bgr;isoforms, erythropoietin receptor, MPL [thrombopoietin receptor], and G-CSF receptor), heterodimers whose subunits each have ligand-binding and effector domains (e.g., PDGF receptor &agr;&bgr;isoform), and multimers having component subunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunits are common to a plurality of receptors. For example, the AIC2B subunit, which cannot bind ligand on its own but includes an intracellular signal transduction domain, is a component of IL-3 and GM-CSF receptors. Many cytokine receptors can be placed into one of four related families on the basis of their structures and functions. Hematopoietic receptors, for example, are characterized by the presence of a domain containing conserved cysteine residues and the WSXWS motif. Additional domains, including protein kinase domains; fibronectin type III domains; and immunoglobulin domains, which are characterized by disulfide-bonded loops, are present in certain hematopoietic receptors. Cytokine receptor structure has been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228 (1991) and Cosman, Cytokine 5:95-106 (1993). It is generally believed that under selective pressure for organisms to acquire new biological functions, new receptor family members arose from duplication of existing receptor genes leading to the existence of multi-gene families. Family members thus contain vestiges of the ancestral gene, and these characteristic features can be exploited in the isolation and identification of additional family members. The cytokine receptor superfamily is subdivided as shown in Table 1. 1 TABLE 1 Cytokine Receptor Superfamily Immunoglobulin family CSF-1 receptor MGF receptor IL-1 receptor PDGF receptor Hematopoietin family erythropoietin receptor G-CSF receptor IL-2 receptor b-subunit IL-3 receptor IL-4 receptor IL-5 receptor IL-6 receptor IL-7 receptor IL-9 receptor GM-CSF receptor a-subunit GM-CSF receptor b-subunit Prolactin receptor CNTF receptor Oncostatin M receptor Leukemia inhibitory factor receptor Growth hormone receptor MPL Leptin receptor TNF receptor family TNF (p80) receptor TNF (p60) receptor TNFR-RP CD27 CD30 CD40 4-1BB OX-40 Fas NGF receptor Other IL-2 receptor &agr;-subunit IL-15 receptor &agr;-subunit IFN-&ggr; receptor
[0041] Cell-surface cytokine receptors are further characterized by the presence of additional domains. These receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues (typically about 21-25 residues), which is commonly flanked by positively charged residues (Lys or Arg). On the opposite end of the protein from the extracellular domain and separated from it by the transmembrane domain is an intracellular domain.
[0042] The novel receptor of the present invention was initially identified by the presence of the conserved WSXWS motif. Analysis of a human cDNA clone encoding human Zcytor5 (SEQ ID NO:1) revealed an open reading frame encoding 422 amino acids (SEQ ID NO:2) or an allelic variant reveals an open reading of 425 amino acid residues, SEQ ID NO:3 and SEQ ID NO:4.
[0043] Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is at least about 0.02 M at pH 7 and the temperature is at least about 60° C. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from testis, including whole testis tissue extracts or testicular cells, such as Sertoli cells, Leydig cells, spermatogonia, or epididymis, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient [Chirgwin et al., Biochemistry 18:52-94 (1979)]. Poly (A)+RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly(A)+RNA using known methods. Polynucleotides encoding Zcytor5 polypeptides are then identified and isolated by, for example, hybridization or PCR.
[0044] Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS:1, 2, 3, 4 represent single alleles of the human and SEQ ID NOs 5 and 6 of the rat Zcytor5 receptors. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
[0045] The present invention further provides counterpart receptors and polynucleotides from other species (“species orthologs”). Of particular interest are Zcytor5 receptors from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate receptors. Species orthologs of the human and macaque Zcytor5 receptors can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the receptor. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A receptor-encoding cDNA can then be isolated-by a variety of methods, such as by probing with a complete or partial human or macaque cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers designed from the sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the receptor. Similar techniques can also be applied to the isolation of genomic clones.
[0046] The polynucleotides of the present invention can be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
[0047] One method for building a synthetic gene requires the initial production of a set of overlapping, complementary oligonucleotides, each of which is between 20 to 60 nucleotides long. The sequences of the strands are planned so that, after annealing, the two end segments of the gene are aligned to give blunt or staggered ends. Each internal section of the gene has complementary 3′ and 5′ terminal extensions that are designed to base pair precisely with an adjacent section. Thus, after the gene is assembled, the only remaining requirement to complete the process is sealing the nicks along the backbones of the two strands with T4 DNA ligase. In addition to the protein coding sequence, synthetic genes can be designed with terminal sequences that facilitate insertion into a restriction endonuclease sites of a cloning vector and other sequences should also be added that contain signals for the proper initiation and termination of transcription and translation. See Glick, Bernard R. and Jack J. Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994), Itakura, K. et al. Synthesis and use of synthetic oligonucleotides. Annu. Rev. Biochem. 53:323-356 (1984), and Climie, S. et al. Chemical synthesis of the thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 87 :633-637 (1990).
[0048] Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of the this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. A region of a protein to which an antibody can bind is defined as an “antigenic epitope”. See for instance, Geysen, H. M. et al., Proc. Natl. Acad Sci. USA 81:3998-4002 (1984).
[0049] As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in the art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J. G. et al. Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.
[0050] Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides defined by SEQ ID NOs:32-37.
[0051] The present invention also provides isolated receptor polypeptides that are substantially identical to the receptor polypeptides of SEQ ID NOs: 2, 4 and 6 and their species orthologs. By “isolated” is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. The term “substantially homologous” is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2, 4, or 7 or their species orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2, 4 or 6 or their species orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty-of 1, and the “blossom 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 2 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as: 1 Total number of identical matches [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] × 100 2 TABLE 2 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4
[0052] Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
[0053] Polynucleotides, generally a cDNA sequence, of the present invention encode the above-described polypeptides. A cDNA sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows. 3 Alanine (Ala) is encoded by GCA, GCC, GCG or GCT; Cysteine (Cys) is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid (Glu) is encoded by GAA or GAG; Phenylalanine (Phe) is encoded by TTC or TTT; Glycine (Gly) is encoded by GGA, GGC, GGG or GGT; Histidine (His) is encoded by CAC or CAT; Isoleucine (Ile) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA, CCC, CCG or CCT; Glutamine (Gln) is encoded by CAA or CAG; Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is encoded by ACA, ACC, ACG or ACT; Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and Tyrosine (Tyr) is encoded by TAC or TAT.
[0054] It is to be recognized that according to the present invention, when a cDNA is claimed as described above, it is understood that what is claimed are both the sense strand, the anti-sense strand, and the DNA as double-stranded having both the sense and anti-sense strand annealed together by their respective hydrogen bonds. Also claimed is the messenger RNA (mRNA) which encodes the polypeptides of the present invention, and which mRNA is encoded by the above-described cDNA. A messenger RNA (mRNA) will encode a polypeptide using the same codons as those defined above, with the exception that each thymine nucleotide (T) is replaced by a uracil nucleotide (U).
[0055] Substantially identical proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 3) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tract, protein A, Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:.3 (1991), glutathione S transferase, Smith and Johnson, Gene 67:31 (1988), or other antigenic epitope or binding domain. See, in general Ford et al., Protein Expression and Purification 2: 95-107 (1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.). 4 TABLE 3 Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine
[0056] Essential amino acids in the receptor polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502 (1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., ligand binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-312 (1992) ; Smith et al., J. Mol. Biol. 224:899-904 (1992); Wlodaver et al., FEBS Lett. 309:59-64 (1992). The identities of essential amino acids can also be inferred from analysis of homologies with related receptors.
[0057] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer, Science 241:53-57 (1988) or Bowie and Sauer, Proc. Natl. Acad. Sci. USA 86:2152-2156. (1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display, e.g., Lowman et al., Biochem. 30:10832-10837 (1991); Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis, Derbyshire et al., Gene 46:145 (1986); Ner et al., DNA 7:127, (1988).
[0058] Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized receptors in host cells. Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below. Mutagenized DNA molecules that encode active receptors or portions thereof (e.g., ligand-binding fragments) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
[0059] Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptides that are substantially homologous to residues SEQ ID NOs:2, 4, 6, 17, 18, or 19or allelic variants thereof and retain the ligand-binding properties of the wild-type receptor.
[0060] The receptor polypeptides of the present invention, including full-length receptors, receptor fragments (e.g. ligand-binding fragments), and fusion polypeptides can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., ibid.
[0061] In general, a DNA sequence encoding a Zcytor5 receptor polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
[0062] To direct a Zcytor5 receptor polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the receptor, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is joined to the Zcytor5 DNA sequence in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
[0063] Cultured mammalian cells are preferred hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al., Cell 14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981), Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al., EMBO J. 1:841-845 (1982), DEAE-dextran mediated transfection, Ausubel et al., eds., Current Protocols in Molecular Biology (John Wiley and Sons, Inc., N.Y., 1987), and liposome-mediated transfection, Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone et al., Focus 15:80 (1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, (1977) and Chinese hamster ovary (e.g. CHO-K1 ; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978,) and the adenovirus major late promoter.
[0064] Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.” A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as “amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.
[0065] Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463, which are incorporated herein by reference. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).
[0066] Fungal cells, including yeast cells, and particularly cells of the genus Saccharomyces, can also be used within the present invention, such as for producing receptor fragments or polypeptide fusions. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465 (1986) and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.
[0067] Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
[0068] Within one aspect of the present invention, a novel receptor is produced by a cultured cell, and the cell is used to screen for ligands for the receptor, including the natural ligand, as well as agonists and antagonists of the natural ligand. To summarize this approach, a cDNA or gene encoding the receptor is combined with other genetic elements required for its expression (e.g., a transcription promoter), and the resulting expression vector is inserted into a host cell. Cells that express the DNA and produce functional receptor are selected and used within a variety of screening systems.
[0069] Mammalian cells suitable for use in expressing Zcytor5 receptors and transducing a receptor-mediated signal include cells that express a &bgr;-subunit, such as the human &bgr;c subunit. In this regard it is generally preferred to employ a cell that is responsive to other cytokines that bind to receptors in the same subfamily, such as IL-3 or GM-CSF, because such cells will contain the requisite signal transduction pathway(s). It is also preferred to use a cell from the same species as the receptor to be expressed. Within a preferred embodiment, the cell is dependent upon an exogenously supplied hematopoietic growth factor for its proliferation. Preferred cell lines of this type are the human TF-1 cell line (ATCC number CRL-2003) and the AML-193 cell line (ATCC number CRL-9589), which are GM-CSF-dependent human leukemic cell lines. In the alternative, suitable host cells can be engineered to produce a &bgr;-subunit (e.g., bc) or other cellular component needed for the desired cellular response. For example, the murine cell line BaF3, Palacios and Steinmetz, Cell 41: 727-734 (1985); Mathey-Prevot et al. , Mol. Cell. Biol. 6: 4133-4135 (1986) or a baby hamster kidney (BHK) cell line can be transfected to express the human bc subunit (also known as KH97) as well as a Zcytor5 receptor. The latter approach is advantageous because cell lines can be engineered to express receptor subunits from any species, thereby overcoming potential limitations arising from species specificity. In the alternative, species orthologs of the human receptor cDNA can be cloned and used within cell lines from the same species, such as a mouse cDNA in the BaF3 cell line. Cell lines that are dependent upon one hematopoietic growth factor, such as GM-CSF, can thus be engineered to become dependent upon a Zcytor5 ligand.
[0070] Cells expressing functional receptor are used within screening assays. A variety of suitable assays are known in the art. These assays are based on the detection of a biological response in a target cell. One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the metabolic breakdown of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65: 55-63 (1983). An alternative assay format uses cells that are further engineered to express a reporter gene. The reporter gene is linked to a promoter element that is responsive to the receptor-linked pathway, and the assay detects activation of transcription of the reporter gene. A preferred promoter element in this regard is a serum response element, or SRE, e.g., Shaw et al., Cell 56:563-572 (1989). A preferred reporter gene is a luciferase gene, de Wet et al., Mol. Cell. Biol. 7:725 (1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:29094-29101 (1994); Schenborn and Goiffin, Promega Notes 41:11 (1993). Luciferase activity assay kits are commercially available from, for example, Promega Corp., Madison, Wis. Target cell lines of this type can be used to screen libraries of chemicals, cell-conditioned culture media, fungal broths, soil samples, water samples, and the like. For example, a bank of cell-conditioned media samples can be assayed on a target cell to identify cells that produce ligand. Positive cells are then used to produce a cDNA library in a mammalian expression vector, which is divided into pools, transfected into host cells, and expressed. Media samples from the transfected cells are then assayed, with subsequent division of pools, re-transfection, subculturing, and re-assay of positive cells to isolate a cloned cDNA encoding the ligand.
[0071] A natural ligand for the Zcytor5 receptor can also be identified by mutagenizing a cell line expressing the receptor and culturing it under conditions that select for autocrine growth. See WIPO publication WO 95/21930. Within a typical procedure, BaF3 cells expressing Zcytor5 and human bc are mutagenized, such as with 2-ethylmethanesulfonate (EMS). The cells are then allowed to recover in the presence of IL-3, then transferred to a culture medium lacking IL-3 and IL-4. Surviving cells are screened for the production of a Zcytor5 ligand, such as by adding soluble receptor to the culture medium or by assaying conditioned media on wild-type BaF3 cells and BaF3 cells expressing the receptor.
[0072] An additional screening approach provided by the present invention includes the use of hybrid receptor polypeptides. These hybrid polypeptides fall into two general classes. It is preferred that the second receptor be a hematopoietic cytokine receptor, such as mpl receptor (Souyri et al., Cell 63: 1137-1147 (1990). The hybrid receptor will further comprise a transmembrane domain, which may be derived from either receptor. A DNA construct encoding the hybrid receptor is then inserted into a host cell. Cells expressing the hybrid receptor are cultured in the presence of a ligand for the binding domain and assayed for a response. This system provides a means for analyzing signal transduction mediated by Zcytor5 while using readily available ligands. This system can also be used to determine if particular cell lines are capable of responding to signals transduced by Zcytor5.
[0073] Cells found to express the ligand are then used to prepare a cDNA library from which the ligand-encoding cDNA can be isolated as disclosed-above. The present invention thus provides, in addition to novel receptor polypeptides, methods for cloning polypeptide ligands for the receptors.
[0074] Compounds identified as receptor agonists are useful for stimulating proliferation and development of target cells in vitro and in vivo. For example, agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and/or development of testis-derived cells in culture. Agonists and antagonists may also prove useful in the study of spermatogenesis and infertility. Antagonists are useful as research reagents for characterizing sites of ligand-receptor interaction. In vivo, receptor agonists may find application in the treatment of male infertility. Antagonists of receptor function may be useful as male contraceptive agents.
[0075] The proposed cytokine binding domain of Zcytor5 appears to be closest to the Interleukin-6 &bgr;-chain or gp130 (29% identity). The ligand for Zcytor5 is probably a member of the Interleukin-6 family of cytokines which at present includes: Interleukins-6, -11, Leukemia Inhibitory Factor, Oncostatin M, Cardiotropin-1 and Ciliary Neurotrophic Factor.
[0076] All Zcytor5 cDNAs isolated thus far do not encode a transmembrane domain nor any recognizable cytoplasmic signaling motifs characteristic of the Class I receptors. Structurally, Zcytor5 bears close similarity to &agr;-subunit of the Ciliary neutrophil Factor receptor (CNTF-R&agr;). It is quite possible Zcytor5 does not have a transmembrane domain form and that the native molecule is phosphatidyl-inositol linked to the cell membrane in a manner similar to CNTF-R&agr;.
[0077] Rebledo et al. (J. Biol. Chem., 272: 4855-4863) provide evidence for the existence of a third component of the Cardiotropin-1 receptor (CT-1R). CT-1R is believed to have a tripartite structure comprised of gp130, gp190 (LIV Receptor &bgr;) and an uncharacterized 45 kDa (CT-1R&agr;) subunit that appears to be linked to the cell surface through a phosphatidyl-inositol linkage. CT-1R &agr;appears to be important for increased sensitivity and specificity of the receptor complex to Cardiotropin-1. The data suggests that Zcytor5 is CT-1R&agr;. Cardiotropin-1 is a member of the Interleukin-6 family in which gp130 and gp190 are members of a tripartite complex is the Ciliary neurotropic Factor receptor. In this receptor complex, CNTF-R&agr; comprises the third receptor subunit and it mediates specificity and high affinity binding of the ligand complex. These functions are similar to the proposed ones for CT-1R&agr;. One might then argue on the basis of “symmetry of nature” that CT-1R &agr; would physically resemble CNTF-R&agr; and that the close structural similarity of Zcytor5 to CNTF-R&agr; would make Zcytor5 a possible candidate for the third subunit of CT-1R. Furthermore, the proposed 45 kDa molecular mass of CT-R&agr; agrees with that of Zcytor5 and the transcripts of CT-1 and Zcytor5 are found in similar tissues. In particular, both transcripts are found in high levels in heart and in skeletal muscles, which is consistent with the observation that ligand and their receptor subunits are often co-expressed in the same tissue.
[0078] Cardiotropin-1 was originally cloned by function as a factor involved in cardiac hypertrophy, an adaptive response of heart muscle to an increased work load. Hypertrophy is characterized by reactivation of genes expressed during fetal heart development and by the accumulation of carsomeric proteins. If Zcytor5 proves to be the subunit that is important in the binding and specificity of Cardiotropin-1 to its receptor, Zcytor5 may prove to be a useful therapeutic antagonist to counteract the hypertrophic response to injury. Cardiotropin-1 has also been shown to promote survival of rat dopaminergic neurons in vitro. An agonist-active soluble receptor may potentially be useful in the treatment of neuronal disorders such as Parkinson's disease.
[0079] Zcytor5 may also be used within diagnostic systems for the detection of circulating levels of ligand. Within a related embodiment, antibodies or other agents that specifically bind to Zcytor5 can be used to detect circulating receptor polypeptides. Elevated or depressed levels of ligand or receptor polypeptides may be indicative of pathological conditions, including cancer.
[0080] Zcytor5 receptor polypeptides can be prepared by expressing a DNA encoding a Zcytor5 polypeptide as described in SEQ ID NO:1, 3 and 5. To direct the export of the receptor domain from the host cell, the receptor DNA is linked to a second DNA segment encoding a secretory peptide. It is believed that amino acid 1-34 or in the alternative amino acid residues 1-30 are secretory peptides of SEQ ID NO:2. For SEQ ID NO:4, it is believed that residues 1-33 or in the alternative 1-29 are secretory peptides. For the rat sequence, it is believed that amino acid residues 1-40 define a secretory peptide. These peptides are generally cleaved after secretion by a mammalian cell. In the alternative, other secretory peptides could be fused to the Zcytor5 polypeptide, such as the t-PA secretory peptide. To facilitate purification of the secreted receptor domain, a C-terminal extension, such as a poly-histidine tag, substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210 (1988); available from Eastman Kodak Co., New Haven, Conn.) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the receptor polypeptide.
[0081] In an alternative approach, a receptor extracellular domain can be expressed as a fusion with immunoglobulin heavy chain constant regions, typically an Fc fragment, which contains two constant region domains and a hinge region but lacks the variable region. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two receptor polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify the cognate ligand from solution, as an in vitro assay tool, to block signals in vitro by specifically titrating out ligand, and as antagonists in vivo by administering them parenterally to bind circulating ligand and clear it from the circulation. To purify ligand, a Zcytor5-Ig chimera is added to a sample containing the ligand (e.g., cell-conditioned culture media or tissue extracts) under conditions that facilitate receptor-ligand binding (typically near-physiological temperature, pH, and ionic strength). The chimera-ligand complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads). The ligand is then eluted using conventional chemical techniques, such as with a salt or pH gradient. In the alternative, the chimera itself can be bound to a solid support, with binding and elution carried out as above. The chimeras may be used in vivo to induce infertility. Chimeras with high binding affinity are administered parenterally (e.g., by intramuscular, subcutaneous or intravenous injection). Circulating molecules bind ligand and are cleared from circulation by normal physiological processes. For use in assays, the chimeras are bound to a support via the Fc region and used in an ELISA format.
[0082] A preferred assay system employing a ligand-binding receptor fragment uses a commercially available biosensor instrument (BIAcore™, Pharmacia Biosensor, Piscataway, N.J.), wherein the receptor fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immonol Methods 145:229-240 (1991) and Cunningham and Wells, J. Mol. Biol. 234:554-563 (1993). A receptor fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If ligand is present in the sample, it will bind to the immobilized receptor polypeptide, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
[0083] Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity, Scatchard, Ann. NY Acad. Sci. 51: 660-672 (1949) and calorimetric assays, Cunningham et al., Science 253:545-548 (1991); Cunningham et al., Science 254:821-825 (1991).
[0084] A receptor ligand-binding polypeptide can also be used for purification of ligand. The receptor polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting media will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration or pH to disrupt ligand-receptor binding.
[0085] Zcytor5 polypeptides can also be used to prepare antibodies that specifically bind to Zcytor5 polypeptides. As used herein, the term “antibodies” includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)2 and Fab fragments, and the like, including genetically engineered antibodies. Antibodies are defined to be specifically binding if they bind to a Zcytor5 polypeptide with a Ka of greater than or equal to 107/M. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, ibid.).
[0086] Zcytor5 polypeptides can also be used to prepare antibodies that specifically bind to Zcytor5 polypeptides. These antibodies can then be used to manufacture anti-idiotypic antibodies. As used herein, the term “antibodies” includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)2 and Fab fragments, and the like, including genetically engineered antibodies. Antibodies are defined to be specifically binding if they bind to a Zcytor5 polypeptide with a Ka of greater than or equal to 107/M. The affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, ibid.).
[0087] Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, (Second Edition) (Cold Spring Harbor, N.Y., 1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Inc., Boca Raton, Fla., 1982). As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a Zcytor5 polypeptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Zcytor5 polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, and sandwich assays.
[0088] As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs and rats with a Zcytor5 polypeptide or a fragment thereof. The immunogenicity of a Zcytor5 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zcytor5 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is “hapten-like”, such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
[0089] As used herein, the term “antibodies” includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab′)2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally “cloaking” them with a human-like surface by replacement of exposed residues, wherein the result is a “veneered” antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
[0090] Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to Zcytor5 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zcytor5 protein or peptide). Genes encoding polypeptides having potential Zcytor5 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display libraries can be screened using the Zcytor5 sequences disclosed herein to identify proteins which bind to Zcytor5. These “binding proteins” which interact with Zcytor5 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as Zcytor5 “antagonists” to block Zcytor5 binding and signal transduction in vitro and in vivo.
[0091] Antibodies can also be generated gene therapy. The animal is administered the DNA or RNA which encodes Zcytor5 or an immunogenic fragment thereof so that cells of the animals are transfected with the nucleic acid and express the protein which in turn elicits an immunogenic response. Antibodies which then are produced by the animal are isolated in the form of polyclonal or monoclonal antibodies.
[0092] Antibodies to Zcytor5 may be used for tagging cells that express the protein, for affinity purification, within diagnostic assays for determining circulating levels of soluble protein polypeptides, and as antagonists to block ligand binding and signal transduction in vitro and in vivo.
[0093] Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Inc., Boca Raton, Fla., 1982). As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. Generally speaking, antibodies against which bind to the claimed Zcytor5 polypeptides can be raised by immunization of animals with a Zcytor5 polypeptide or a fragment thereof. The immunogenicity of a Zcytor5 polypeptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Zcytor5 polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, and sandwich assays.
[0094] Antibodies to Zcytor5 are may be used for tagging cells that express the receptor, for affinity purification, within diagnostic assays for determining circulating levels of soluble receptor polypeptides, and as antagonists to block ligand binding and signal transduction in vitro and in vivo.
Uses[0095] The tissue specificity of Zcytor5 expression suggests that Zcytor5 may be a receptor for growth and/or maintenance factor in the thyroid heart and skeletal muscle. Zcytor5 could therefor be used to down regulate the effects of the factor by administering soluble Zcytor5 to the patient. For example the soluble receptor could be used to lessen the effect of cardiotrophin-1 on cardiac pathologies. Thus preventing enlargement of the heart due to heart disease. Zcytor5 could also be used as a diagnostic to test for the presence of cardiotrophin-1 in the blood. Furthermore, Zcytor5 can be used to discover other possible ligands which would bind to Zcytor5.
[0096] The present invention also provides reagents which will find use in diagnostic applications. For example, the Zcytor5 gene. A probe comprising the Zcytor5 DNA or RNA or a subsequence thereof can be used to determine if the Zcytor5 gene is present on chromosome 1 or if a mutation has occurred.
[0097] Antibodies to Zcytor5 could be used to purify Zcytor5 and as a therapeutic to modulate the effect of the Zcytor5 ligand. The anti-idiotypic antibody to Zcytor5 could be used to purify the ligand of Zcytor5 and the administration of the anti-idiotypic antibody could be used to modulate the effect of the Zcytor5 ligand.
[0098] The invention is further illustrated by the following non-limiting examples.
EXAMPLE 1 Cloning of Human Zcytor 5[0099] Human Zcytor5 was identified from expressed sequence tag (EST) 698365 (SEQ ID NO:7) identified in an EST database. The cDNA containing EST 698365 was obtained from Incyte Pharmaceuticals, Inc. as dried DNA. Upon reconstitution in water, the cDNA was transfected into E. coli strain DH10B. The plasmid was designated pSL8365. The EST in plasmid pSL8365 was sequenced, revealing an insert of 952 bp.
[0100] The GENE TRAPPER® cDNA positive selection system (Life Technologies, Gaithersburg, Md.) employing oligonucleotide ZC11,286 (SEQ ID NO:8) was used to isolate the plasmid Hzcytor5-9 from a human lung cDNA library (obtained from Life Technologies Inc., Gaithersburg, Md.) in accordance with the manufacturer's directions. Hzcytor5-9 extended the sequence of pSL8365 by 459 bp. The sequence present in Hzcytor5-9 allowed the isolation of an overlapping EST No. 485212 (SEQ ID NO:9), which extended the open reading frame of Hzcytor5-9 by a further 33 codons.
[0101] A cDNA encoding full-length Zcytor5 was isolated from a human testis cDNA library. (See Example 2 for the preparation of the human cDNA testis library.) The library was comprised of eighty pools of plasmid DNA, each pool comprised of 10,000 independent recombinants. The presence of Zcytor5 cDNA in each library pool was determined by PCR employing primers ZC11,663 (SEQ ID NO:10) and ZC12,212 (SEQ ID NO:11). PCR was carried out using AMPLITAQ® DNA polymerase (Perkin-Elmer) in buffer conditions recommended by the supplier. The amplification was carried out at 94° C. for 1 minute followed by 30 cycles, each cycle consisting of 20 seconds at 94° C., 1 minute at 66° C. and 7 minutes at 74° C. Five cDNA pools were found to be positive for the 420 bp PCR product by agarose gel electrophoresis.
[0102] Plasmid DNA from one positive library pool was electrophoresed into DH10B cells and plated. Colony lifts were prepared using Hybond-N filters (Amersham; Arlington Heights, Ill.) according to the procedure provided by the manufacturer. Following denaturation and neutralization, DNA was cross-linked onto the filters with 1,200 &mgr;Joules of UV energy in a STRATALINKER® (Stratagene Cloning Systems). Cell debris was removed-by several washes in 0.25×standard sodium citrate (SSC), 0.25% sodium dodecyl sulfate (SDS) and 1 mM EDTA at 65° C. The filters were then pre-hybridized overnight at 65° C. in EXPRESSHYB® solution (Clontech) with 1 mg/ml heat denatured salmon sperm DNA. Colonies positive for Zcytor5 were identified by hybridization with a probe that was generated from EST 484212 (SEQ ID NO:9) cDNA employing PCR primers ZC11,663 (SEQ ID NO:10) and ZC12,212 (SEQ ID NO:11). The PCR product probe was purified by agarose gel electrophoresis. 100 ng of the probe was labeled with 32p dCTP using the MULTI-PRIME® DNA labeling system (Amersham). Unincorporated label was removed with a NUCTRAP® column (Stratagene). Probe hybridization was carried out overnight at 65° C. in EXPRESSHYB® solution at a probe concentration of 1×106 cpm/ml. The filters were washed at 65° C. in a wash buffer containing 0.25×SSC, 0.25 SDS and 1 mM EDTA.
[0103] Three positive signals were identified and were subjected to colony purification via a second round of filter hybridization. Sequence analysis of one positive clone, SEQ ID NO:3 was found to be full length human Zcytor5. Sequencing of a several overlapping clones revealed a second full-length sequence SEQ ID NO:1 which is an allelic variant of SEQ ID NO:3.
EXAMPLE 2 Construction of the Human Testis cDNA Library[0104] Fourteen &mgr;l of poly d(T)—selected poly (A)+human testis mRNA (Clontech) at a concentration of 1.0 &mgr;g/&mgr;l was mixed with 2 &mgr;l of 20 pmole/&mgr;l first strand primer ZC2938 (SEQ ID NO:12 ) containing an Sst I restriction site. The mixture was heated at 65° C. for 4 minutes and cooled by chilling on ice. First strand cDNA synthesis was initiated by the addition of 8 &mgr;l of 250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15 mM MgCl2 (5×SUPERSCRIPT™ buffer; GIECO BRL), 4 &mgr;l of 100 mM dithiothreitol (DTT) and 2 &mgr;l of a deoxynucleotide triphosphate solution containing 10 mM each of DATP, dGTP, dTTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology Inc.) to the RNA-primer mixture. The reaction mixture was incubated at 45° C. for 4 minutes followed by the addition of 10 &mgr;l of 200 U/&mgr;l RNase H− reverse transcriptase (GIBCO BRL). The efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 &mgr;Ci of 32P-&agr;dCTP to a 10 &mgr;l aliquot of the reaction mixture to label the reaction for analysis. The reactions were incubated at 45° C. for 1 hour followed by an incubation at 50° C. for 15 minutes. Unincorporated 32P-&agr;dCTP in the labeled reaction was removed by chromatography on a 400 pore size gel filtration column (CHROMA SPIN+TE-400™; Clontech Laboratories Inc.). Unincorporated nucleotides in the unlabeled first strand reaction were removed by twice precipitating the cDNA in the presence of 10 &mgr;g of glycogen carrier, 2.5 M ammonium acetate and 2.5 volume ethanol. The unlabeled cDNA was resuspended in 50 &mgr;l water for use in second strand synthesis. The length of the labeled first strand cDNA was determined by agarose gel electrophoresis.
[0105] Second strand synthesis was performed on first strand cDNA under conditions that promoted first strand priming of second strand synthesis resulting in DNA hairpin formation. The reaction mixture was assembled at room temperature and was comprised of 66 &mgr;l of the unlabeled first strand cDNA, 20 &mgr;l of 5× polymerase I buffer (100 mM Tris: HCl, pH 7.4, 500 mM KCl, 25 mM MgCl2, 50 mM (NH4)2SO4), 1 &mgr;l of 100 mM DTT, 1 &mgr;l of a solution containing 20 mM of each deoxynucleotide triphosphate, 3 &mgr;l of 5 mM &bgr;-NAD, 1 &mgr;l of 4 U/&mgr;l of E. coli DNA ligase (New England Biolabs Inc., Beverly, Mass.) and 5 &mgr;l of 10 U/&mgr;l E. coli DNA polymerase I (New England Biolabs, Inc.). The reaction was incubated at room temperature for 5 minutes followed by the addition of 2 &mgr;l of 2.2 U/&mgr;l RNase H (GIBCO BRL). A parallel reaction in which a 10 &mgr;l aliquot of the second strand synthesis mixture was labeled by the addition of 10 &mgr;Ci 32P-&agr;dCTP was used to monitor the efficiency of second strand synthesis. The reactions were incubated at 15° C. for two hours followed by a 15 minute incubation at room temperature. Unincorporated 32 P-&agr;dCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Inc.) before analysis by agarose gel electrophoresis. The unlabeled reaction was terminated by two extractions with phenol/chloroform and a chloroform extraction followed by ethanol precipitation in the presence of 2.5 M ammonium acetate.
[0106] The single-stranded DNA of the hairpin structure was cleaved using mung bean nuclease. The reaction mixture contained 100 &mgr;l of second strand cDNA, 20 &mgr;l of 10×mung bean nuclease buffer (Stratagene Cloning Systems, La Jolla, Calif.), 16 &mgr;l of 100 mM DTT, 51.5 &mgr;l of water and 12.5 &mgr;l of a 1:10 dilution of mung bean nuclease (Promega Corp.; final concentration 10.5 U/&mgr;l) in mung bean nuclease dilution buffer. The reaction was incubated at 37° C. for 15 minutes. The reaction was terminated by the addition of 20 &mgr;l of 1 M Tris: HCl, pH 8.0 followed by sequential phenol/chloroform and chloroform extractions as described above. Following the extractions, the DNA was precipitated in ethanol and resuspended in water.
[0107] The resuspended cDNA was blunt-ended with T4 DNA polymerase. The cDNA, which was resuspended in 138 &mgr;l of water, was mixed with 40 &mgr;l of 5×T4 DNA polymerase buffer (250 mM Tris: HCl, PH 8.0, 250 mM KCl, 25 mM MgCl2), 3 &mgr;l 0.1 M DTT, 5 &mgr;l of a solution containing 10 mM of each deoxynucleotide triphosphate and 4 &mgr;l of 1 U/&mgr;l T4 DNA polymerase (Boehringer Mannheim Corp., Indianapolis, Ind.). After incubation of 1 hour at 10° C., the reaction was terminated by the addition of 10 &mgr;l of 0.5 M EDTA followed by serial phenol/chloroform and chloroform extractions as described above. The DNA was chromatographed through a 400 pore size gel filtration column (Clontech Laboratories Inc. Palo Alto, Calif.) to remove trace levels of protein and to remove short cDNAs less than about 400 bp in length. The DNA was ethanol precipitated in the presence of 12 &mgr;g glycogen carrier and 2.5 M ammonium acetate and was resuspended in 10 &mgr;l of water. Based on the incorporation of 32P-&agr;dCTP, the yield of cDNA was estimated to be about 2 &mgr;g from a starting template of 12.5 &mgr;g.
[0108] Eco RI adapters were ligated onto the 5′ ends of the cDNA to enable cloning into a lambda phage vector. A 10 &mgr;l aliquot of cDNA (containing about 2 &mgr;g of cDNA) and 11 &mgr;l of 65 pmole/&mgr;l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.) were mixed with 3 &mgr;l 10× ligase buffer (Promega Corp.), 3 &mgr;l 10 mM ATP and 3 &mgr;l of 15 U/&mgr;l T4 DNA ligase (Promega Corp.). The reaction was incubated overnight (about 18 hours) at 12.5° C. The reaction was terminated by the addition of 150 &mgr;l of water and 10 &mgr;l of 3 M Na acetate, followed by incubation at 65° C. for 30 minutes. After incubation, the cDNA was extracted with phenol/chloroform and chloroform as described above and precipitated in the presence of 2.5 M ammonium acetate and 1.2 volume of isopropanol. Following centrifugation, the cDNA pellet was washed with 70% ethanol, air dried and resuspended in 89 &mgr;l water.
[0109] To facilitate the directional cloning of the cDNA into a lambda phage vector, the cDNA was digested with Sst-I resulting in a cDNA having 5′ Eco RI and 3′ Sst-I cohesive ends. The Sst-I restriction site at the 3′ end of the cDNA had been previously introduced through primer ZC2938 (SEQ ID NO:12). Restriction enzyme digestion was carried out in a reaction containing 89 &mgr;l of cDNA described above, 10 &mgr;l of 6 mM Tris: HCl, 6 mM MgCl2, 150 mM NaCl, 1 mM DTT (10 ×D buffer; Promega Corp., Madison, Wis.) and 1 &mgr;l of 12 U/&mgr;l Not I (Promega Corp.). Digestion was carried out at 37° C. for 1 hour. The reaction was terminated by serial phenol/chloroform and chloroform extractions. The cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 20 &mgr;l of 1× gel loading buffer (10 mM Tris: HCl, pH 8.0, 1 mM EDTA, 5% glycerol and 0.125% bromphenol blue).
[0110] The resuspended cDNA was heated to 65° C. for 5 minutes, cooled on ice and electrophoresed on a 0.8% low melt agarose gel ( SEA PLAQUE GTG™ low melt agarose; FMC Corp.). Unincorporated adapters and cDNA below 1.6 kb in length were excised from the gel. The electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane of origin. The area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined. A 300 &mgr;l aliquot of water, approximately three times the volume of the gel slice, was added to the tube. The agarose was then melted by heating to 65° C. for 15 minutes. Following equilibration of the sample to 42° C., 10 &mgr;l of 1 U/&mgr;l &bgr;-agarose I ( New England Biolabs, Inc.) was added, and the mixture was incubated for 90 minutes to digest the agarose. After incubation, 40 &mgr;l of 3 M Na acetate was added to the sample, and the mixture was incubated on ice for 15 minutes. The sample was centrifuged at 14,000 ×g for 15 minutes at room temperature to remove undigested agarose. The cDNA in the supernatant was ethanol precipitated, washed in 70% ethanol, air-dried and resuspended in 37 &mgr;l of water for the kinase reaction to phosphorylate the ligated Eco RI adapters.
[0111] To the 37 &mgr;l cDNA solution described above was added 10 &mgr;l of 10×ligase buffer (Stratagene Cloning Systems), and the mixture was heated to 65° C. for 5 minutes. The mixture was cooled on ice, and 5 &mgr;l of 10 mM ATP and 3 &mgr;l of 10 U/&mgr;l of T4 polynucleotide kinase (Stratagene Cloning Systems) were added. The reaction was incubated at 37° C. for 45 minutes and was terminated by heating to 65° C. for 10 minutes followed by serial extractions with phenol/chloroform and chloroform. The phosphorylated cDNA was ethanol precipitated in the presence of 2.5 M ammonium acetate, washed with 70%. ethanol, air dried and resuspended in 12.5 &mgr;l water. The concentration of the phosphorylated cDNA was estimated to be about 40 fmole/&mgr;l.
EXAMPLE 3 Northern Blot Analysis of Human Zcytor5[0112] A 300 bp double stranded DNA probe for Northern analysis was prepared from pSL1034 by PCR using oligonucleotide primers ZC 10,787 (SEQ ID NO:13) and ZC 11,097 (SEQ ID NO:14). The 300 bp PCR fragment was gel-purified using a QIAQUICK® purification kit (Qiagen Inc., Chatsworth, Calif.) and random-primer labeled using a MULTIPRIME® kit (Amersham Corp.). Labeled cDNA was purified from free counts using a- Stratagene push column. Human multiple tissue Northern blots (Clontech Laboratories) and a human fetal tissue Northern blot (Clontech Laboratories) were pre-hybridized for three hours at 68° C. using EXPRESSHYB hybridization solution (Clontech Laboratories). The 32 P-labeled cDNA probe was then added to 10 mls of fresh hybridization solution at 106cpm/ml overnight at 68° C. The-blots were washed several times at room temperature in wash solution containing 2×SSC, 0.05% SDS, then with continuous agitation for 40 min at room temperature. The blots were then washed in 0.1×SSC, 0.1% SDS at 50° C. for 40 min with one change of wash solution.
[0113] A single transcript of ˜2.3 kb was detected after exposure to film. In the multiple tissue blots (MTN, MTN II and MTN III; Clontech Laboratories) the transcript was present in highest abundance in placenta, thyroid, heart and skeletal muscle with lower levels in prostate and trachea. Trace mRNA levels were found in kidney, pancreas, testis, small intestine, colon, lymph node, adrenal cortex and bone marrow.
EXAMPLE 4 Chromosomal Assignment and Placement of Human Zcytor-5[0114] Zcytor5 was mapped to chromosome 19 using the commercially available version of the “Stanford G3 Radiation Hybrid Mapping Panel” (Research Genetics, Inc., Huntsville, Ala.). The “Stanford G3 RH Panel” contains PCRable DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient). A publicly available WWW server (http://shgc- www.stanford.edu) allows chromosomal localization of markers.
[0115] For the mapping of Zcytor5 with the “Stanford G3 RH Panel”, 20 &mgr;l reactions were set up in a PCRable 96-well microtiter plate (Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2 &mgr;l 10×KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 &mgr;l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 &mgr;l sense primer, (SEQ ID NO:13) 5′ TAT GGC CAG GAC AAC ACA 3′, 1 &mgr;l antisense primer, (SEQ ID NO:14), 5′ ATA GGG CGT AAA GAG AGC 3′, 2 &mgr;l “RediLoad” (Research Genetics, Inc., Huntsville, Ala.), 0.4 &mgr;l 50×Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x &mgr;l ddH20 for a total volume of 20 &mgr;l. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95° C., 35 cycles of a 1 minute denaturation at 95° C., 1 minute annealing at 66° C. and 1.5 minute extension at 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, Md.).
[0116] The results showed linkage of Zcytor5 to the framework marker WI-7289 with a LOD score of >10 and at a distance of 14.67 cR—10000 from the marker. The use of surrounding markers positions Zcytor5 in the 19p13.1-p11 region on the integrated LDB chromosome 19 map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.genetics. soton.ac.uk/public13 html/)
EXAMPLE 5 Cloning of the Rat Zcytor5 Gene[0117] Rat Zcytor5 cDNA encoding Zcytor5 was isolated from an amplified Rat testis cDNA library with a probe that was generated by primers ZC12212 (SEQ ID NO: 11) and ZC10785 (SEQ ID NO:15) and 10 ng of plasmid pSL85212 as a template obtained from cDNA containing EST 698365 as described in Example 1. The probe was prepared by PCR by combining 1 &mgr;l containing 10 ng of pSL85212, 1 &mgr;l of ZC12212 having a concentration of 20 pmole/&mgr;l, 1 &mgr;l of ZC10785 having a concentration of 20 pmole/&mgr;l, 0.5 &mgr;l of dNTP having a concentration of 20 mM of dATP, dGTP, dCTP and dTTP, 5 &mgr;l of 10×Klentaq polymerase buffer (Clontech) 5 &mgr;l Klentaq DNA polymerase (Clontech) and 39.5 &mgr;l water. The amplification was carried out at 94° C. for 1 minute followed by 30 cycles, each cycle consisting of 15 seconds at 95° C., 20 seconds at 62° C. and 1 minutes at 68° C. The reaction had a final incubation at 68° C. for 10 minutes.
[0118] The resulting PCR product was diluted 1:100 with water. Four &mgr;l of the diluted PCR product was re-amplified using the above-described conditions and the resultant PCR product was further purified by electrophoresis on low-melt agarose gel. The DNA probe was recovered from low-melt gel by digestion with &bgr;-Agarose I digestion. The rat Zcytor5 gene was then cloned from a rat testis library which was constructed as described below in Example 6.
[0119] In cloning the rat Zcytor5 gene, the library was first amplified by plating 3.106 plaque forming units (pfu) from the previously constructed primary library onto 98 150 mm NZY plates. Ten ml of serum medium was added to each plate and was incubated for several hours at room temperature. Following incubation, the phage lysates were collected and pooled to yield the amplified phage library.
[0120] 1.5 million pfus from the amplified rat testis cDNA library were plated onto 150 mm NZY plates at a density of 40,000 pfu/plate on XL-1 Blue MRF′ host cells. Following incubation at 37° C. overnight, filter lifts were made using HYBOND-N™ membranes (Amersham), according to the procedures provided by the manufacturer. The filters were processed by denaturation in solution containing 1.5 M NaCl and 0.5 M NaOH for 8 minutes at room temperature. The filters were neutralized in 0.5 M Tris: HCl, pH 7.2 for 5 minutes. Phage DNA was fixed onto the filters with 1,200 &mgr;Joules of UV energy in a UV Cross-linker (Stratagene). The filters were then washed with 0.25×SSC at 70° C. to remove excess cellular debris. Filter pre-hybridization was carried out in a hybridization solution containing 5×SSC, 5×Denhardt solution, 0.2% SDS, 1 mM EDTA and heat denatured sheared salmon-sperm DNA at a final concentration of 100 &mgr;g/ml for 72 hours at 60° C.
[0121] 75 ng of probe DNA was labeled with 32p-dCTP using a MEGAPRIME® labeling kit (Amersham) and was purified with a NUCTRAP® column (Stratagene). The labeled probe was heat-denatured and added to fresh hybridization solution at a concentration of 1.5×106 cpm/ml. Into this solution were also added the filters containing the phage particles. Hybridization of the probes to the phage-containing filters was completed overnight at 45° C. Following hybridization, the filters were washed in a solution containing 0.25×SSC, 0.25% SDS and 1 mM EDTA at 50° C. The washed filters were autoradiographed for 72 hours at −70° C. with intensifying screens. Examination of the autoradiographs revealed multiple regions that hybridized with the labeled probe. Agar plugs were picked from 56 regions for plaque purification. Of the positive signals, eleven produce positive phagemids following secondary and tertiary hybridization screens. The plasmids within the positive phagemids were recovered using the EXASSIT/SOLR™ system according to the manufacturer's specifications. A clone designated pSLRatR5-1 was sequenced and found to encode full length Rat Zcytor5 (SEQ ID NO:5)
EXAMPLE 6 Production of Rat Testis cDNA library[0122] The rat first strand cDNA reaction contained 10 &mgr;l of rat testis poly d(T)-selected poly (A)+mRNA (Clontech, Palo Alto, Calif.) at a concentration of 1.0 &mgr;g/&mgr;l, and 2 &mgr;l of 20 pmole/&mgr;L first strand primer ZC6091 (SEQ ID NO:16) containing an Xho I restriction site. The mixture was heated at 70° C. for 4 minutes and cooled by chilling on ice. First strand cDNA synthesis was initiated by the addition of 8 &mgr;l of first strand buffer (5×SUPERSCRIPT™ buffer; Life Technologies, Gaithersburg, Md.), 4 &mgr;l of 100 mM dithiothreitol, and 2 &mgr;l of a deoxynucleotide triphosphate solution containing 10 mM each of DATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, N.J.) to the RNA-primer mixture. The reaction mixture was incubated at 45° C. for 2 minutes, followed by the addition of 10 &mgr;l of 200 U/&mgr;l RNase H- reverse transcriptase (SUPERSCRIPT II®; Life Technologies). The efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 &mgr;Ci of 32P-&agr;dCTP 5 &mgr;l aliquot from one of the reaction mixtures to label he reaction for analysis. The reactions were incubated at 45° C. for 1 hour followed by an incubation at 50° C. for 10 minutes. Unincorporated 32P-&agr;dCTP in the labeled reaction was removed by chromatography on a 400 pore size gel filtration column (Clontech). The unincorporated nucleotides and primers in the unlabeled first strand retains were removed by chromatography on 400 pore size gel filtration column (Clontech). The length of labeled first strand cDNA was determined by agarose gel electrophoresis.
[0123] The second strand reaction contained 102 &mgr;l of the unlabeled first strand cDNA, 30 &mgr;l of 5×polymerase I buffer (125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl2, 50 mM (NH4) 2SO4)), 2 &mgr;l of 100 mM dithiothreitol, 3 &mgr;l of a solution containing 10 mM of each deoxynucleotide triphosphate, 5 &mgr;l of 5 mM &bgr;-NAD, 2 &mgr;l of 3 U/&mgr;l E. coli DNA ligase (New England Biolabs), 5 &mgr;l of 10 U/&mgr;l E. coli DNA polymerase I (New England Biolabs), and 1.5 &mgr;l of 2 U/ &mgr;l RNase H (Life Technologies). A 10 &mgr;l aliquot from one of the second strand synthesis reactions was labeled by the addition of 10 &mgr;Ci 32P-&agr;dCTP to monitor the efficiency of second strand synthesis. The reactions were incubated at 16° C. for two hours, followed by the addition of 10 &mgr;l T4 DNA polymerase (10 U/&mgr;l, Boerhinger Mannheim) and incubated for an additional 5 minutes at 16° C. Unincorporated 32P-&agr;dCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column (Clontech) before analysis by agarose gel electrophoresis. The unlabeled was terminated by the addition of 20 &mgr;l 0.5 EDTA and extraction with phenol/chloroform and chloroform followed by ethanol precipitation in the presence of 2.5 M ammonium acetate. The yield of cDNA was estimated to be approximately 2 &mgr;g from starting mRNA template of 10 &mgr;g.
[0124] Eco RI adapters were ligated onto the 5′ ends of the cDNA described above to enable cloning into an expression vector. A 10.5 &mgr;l aliquot of cDNA (˜2 &mgr;g) and 5 &mgr;l of 65 pmole/&mgr;l of Eco RI adapter (Pharmacia LKB Biotechnology Inc.) were mixed with 2.5 &mgr;l 10×ligase buffer 66 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2.5 &mgr;l of 10 mM ATP and 1 &mgr;l of 15 U/&mgr;l T4 DNA ligase (Promega Corp., Madison, Wis.). The reaction was incubated overnight (˜12 hours) at 12° C. The reaction was terminated by incubation at 70° C. for 20 minutes. After incubation, the reaction was cooled to 37° C. To the reaction was added 2.5 &mgr;l 10 mm ATP and 3 &mgr;l 10 U/&mgr;l T4 polynucleotide kinase (Life Technologies) to phosphorylate the ligated Eco RI adapters.
[0125] To facilitate the directional cloning of the cDNA into an expression vector, the cDNA was digested with Xho I, resulting in a cDNA having a 5′ Eco RI cohesive end and a 3′ Xho I cohesive end. The Xho I restriction site at the 3′ end of the cDNA had been previously introduced using the ZC6091 primer (SEQ ID NO:3). Restriction enzyme digestion was carried out in a reaction mixture containing 25 &mgr;l of cDNA described above, 15 &mgr;l of 10×H Buffer (Boehringer Mannheim), 109 &mgr;l H2O, and 1.0 &mgr;l of 40 U/&mgr;l Xho I (Boehringer Mannheim). Digestion was carried out at 37° C. for 40 minutes. The reaction was terminated by incubation at 65° C for 10 minutes and chromatography through a 400 pore size gel filtration column (Clontech).
[0126] The cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 20 &mgr;l of 1×gel loading buffer (10 mM Tris:HCl, pH 8.0, 1 mM EDTA, 5% glycerol and 0.125% bromphenol blue). The resuspended cDNA was heated to 65° C. for 5 minutes, cooled on ice and electrophoresed on a 0.8% low melt agarose gel. The contaminating adapters and cDNA below 0.6 Kb in length were excised from the gel. The electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin. The area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined. An aliquot of water approximately three times the volume of the gel slice (300 &mgr;l) and 35 &mgr;l 10×&bgr;-agarose I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65° C. for 15 minutes. Following equilibration of the sample to 45° C., 3 &mgr;l of 1 U/&mgr;l &bgr;-agarose I (New England Biolabs) was added, and the mixture was incubated for 60 minutes at 45° C. to digest the agarose. After incubation, 40 &mgr;l of 3 M Na acetate was added to the sample, and the mixture was incubated on ice for 15 minutes. The sample was centrifuged at 14,000 ×g for 15 minutes at room temperature to remove undigested agarose. The cDNA was ethanol precipitated, washed in 70% ethanol, air-dried and resuspended in 10 &mgr;l water.
[0127] The resulting cDNA was cloned into the lambda phage vector &lgr;ZapII that was predigested with Eco RI and Xho I and dephosphorylated (Stratagene Cloning Systems, La Jolla, Calif.). Ligation of the cDNA to the &lgr;ZapII vector was carried out in a reaction mixture containing 1.0 &mgr;l of prepared vector, 1.0 &mgr;l of rat testis cDNA, 1.0 &mgr;l 10×Ligase Buffer (Promega), 1.0 &mgr;l of 10 mM ATP, 5 &mgr;l water, and 1.0 &mgr;l of T4 DNA Ligase at 15 units/ml (Promega). The ligation mixture was incubated at 5° C.-15° C. overnight in a temperature gradient. After incubation, the ligation mixture was packaged into phage using GIGPACK III GOLD packaging extract (Stratagene Cloning Systems) and the resulting library was titered according to the manufacturer's specifications.
Claims
1. An isolated polynucleotide which encodes a mammalian polypeptide, said polypeptide being comprised of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NOs: 17-31.
2. The isolated polynucleotide of claim 1 wherein said polynucleotide is a DNA sequence.
3. The isolated polynucleotide of claim 1 wherein said polynucleotide is an RNA sequence.
4. An expression vector comprising the following operably linked elements:
- a transcription promoter;
- a DNA segment encoding a mammalian polypeptide, said polypeptide being comprised of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NOs: 17-37; and
- a transcription terminator.
5. An isolated polypeptide said polypeptide being comprised of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NOs: 17-31.
6. A peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zcytor5 polypeptide.
7. A polypeptide of claim 6 wherein the polypeptide has amino acid sequence of at least 15 amino acid residues.
8. The polypeptide of claim 7 wherein said polypeptide is selected from the group of polypeptide consisting of the amino acid sequences of SEQ ID NOs: 32-37.
9. An antibody which specifically binds to an epitope-binding sequence of a Zcytor5 polypeptide.
10. An antibody of claim 9 wherein said antibody binds to a polypeptide comprised of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO:6, and SEQ ID NOs: 17-37.
11. An anti-idiotypic antibody of an antibody of claims 9 or 10.
12. A method for producing an antibody which binds to a Zcytor5 polypeptide comprising inoculating an animal with an epitope-bearing amino acid sequence of Zcytor5 polypeptide under conditions wherein said animal produces antibodies which bind to the Zcytor5 polypeptide; and
- isolating said antibodies.
Type: Application
Filed: Jun 13, 2001
Publication Date: Apr 18, 2002
Applicant: ZymoGenetics, Inc.
Inventors: Si Lok (Seattle, WA), Scott R. Presnell (Seattle, WA), Anna C. Jelmberg (Issaquah, WA), Teresa Gilbert (Auburn, WA), Theodore E. Whitmore (Redmond, WA), Donald C. Foster (Lake Forest Park, WA), Robyn L. Adams (Bellevue, WA), Joyce M. Lehner (Seattle, WA)
Application Number: 09880578
International Classification: C07K014/52; C07H021/04; C12P021/02; C12N005/06;
