Novel Carboxylesterase Nucleic Acid Molecules, Proteins and Uses Thereof
The present invention relates to arthropod esterase proteins; to arthropod esterase nucleic acid molecules, including those that encode such esterase proteins; to antibodies raised against such esterase proteins; and to other compounds that inhibit arthropod esterase activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to protect animals from hematophagous arthropod infestation.
This application is a Continuation of co-pending U.S. patent application Ser. No. 10/678,521, filed Oct. 2, 2003; which is a Divisional of U.S. patent application Ser. No. 09/403,942, filed May 2, 2000, and issued as U.S. Pat. No. 6,664,090; which is a 371 filing of International Patent Application No. PCT/US97/20598, filed Nov. 10, 1997, which is a continuation-in-part of U.S. Ser. No. 08/747,221, filed Nov. 12, 1996, which issued as U.S. Pat. No. 6,063,610 on May 16, 2000; all of which are entitled “NOVEL CARBOXYLESTERASE NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF” and are incorporated herein by reference.
FIELD OF THE INVENTIONThe present invention relates to arthropod esterase nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, and/or other inhibitors, as well as their use to protect an animal from hematophagous arthropod infestation.
BACKGROUND OF THE INVENTIONHematophagous arthropod infestation of animals is a health and economic concern because hematophagous arthropods are known to cause and/or transmit a variety of diseases. Hematophagous arthropods directly cause a variety of diseases, including allergies, and also carry a variety of infectious agents including, but not limited to, endoparasites (e.g., nematodes, cestodes, trematodes and protozoa), bacteria and viruses. In particular, the bites of hematophagous arthropods are a problem for animals maintained as pets because the infestation becomes a source of annoyance not only for the pet but also for the pet owner who may find his or her home generally contaminated with insects. As such, hematophagous arthropods are a problem not only when they are on an animal but also when they are in the general environment of the animal.
Bites from hematophagous arthropods are a particular problem because they not only can lead to disease transmission but also can cause a hypersensitive response in animals which is manifested as disease. For example, bites from fleas can cause an allergic disease called flea allergic (or allergy) dermatitis (FAD). A hypersensitive response in animals typically results in localized tissue inflammation and damage, causing substantial discomfort to the animal.
The medical importance of arthropod infestation has prompted the development of reagents capable of controlling arthropod infestation. Commonly encountered methods to control arthropod infestation are generally focused on use of insecticides. While some of these products are efficacious, most, at best, offer protection of a very limited duration. Furthermore, many of the methods are often not successful in reducing arthropod populations. In particular, insecticides have been used to prevent hematophagous arthropod infestation of animals by adding such insecticides to shampoos, powders, collars, sprays, foggers and liquid bath treatments (i.e., dips). Reduction of hematophagous arthropod infestation on the pet has been unsuccessful for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required); (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and (3) the emergence of hematophagous arthropod populations resistant to the prescribed dose of pesticide. However, hematophagous arthropod populations have been found to become resistant to insecticides.
Prior investigators have described insect carboxylesterase (CE) protein biochemistry, for example, Chen et al., Insect Biochem. Molec. Biol., 24:347-355, 1994; Whyard et al., Biochemical Genetics, 32:924, 1994 and Argentine et al., Insect Biochem. Molec Biol, 25:621-630, 1995. Other investigators have disclosed certain insect CE amino acid sequences, for example, Mouches et al., Proc Natl Acad Sci USA, 87:2574-2578, 1990 and Cooke et al., Proc Natl Acad Sci USA, 86:1426-1430, 1989, and nucleic acid sequence (Vaughn et al., J. Biol. Chem., 270:17044-17049, 1995).
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- Prior investigators have described certain insect juvenile hormone esterase (JHE) nucleic acid and amino acid sequences: for example, sequence for Heliothis virescens is disclosed by Hanzlik et al., J. Biol. Chem., 264:12419-12425, 1989; Eldridge et al., App Environ Microbiol, 58:1583-1591, 1992; Bonning et al., Insect Biochem. Molec. Biol., 22:453-458, 1992; Bonning et al., Natural and Engineered Pest Management Agents, pp. 368-383, 1994 and Harshman et al., Insect Biochem. Molec. Biol, 24:671-676, 1994; sequence for Manduca sexta is disclosed by Vankatesh et al., J Biol Chem, 265:21727-21732, 1990; sequence for Trichoplusia ni is disclosed by Venkatararnan et al., Dev. Genet., 15:391-400, 1994 and Jones et al., Biochem. J, 302:827-835, 1994; and sequence for Lymantria dispar is disclosed by Valaitis, Insect Biochem. Molec. Biol., 22:639-648, 1992.
Identification of an esterase of the present invention is unexpected, however, because even the most similar nucleic acid sequence identified by previous investigators could not be used to identify an esterase of the present invention. In addition, identification of an esterase protein of the present invention is unexpected because a protein fraction from flea prepupal larvae that was obtained by monitoring for serine protease activity surprisingly also contained esterase proteins of the present invention.
In summary, there remains a need to develop a reagent and a method to protect animals or plants from hematophagous arthropod infestation.
SUMMARY OF THE INVENTIONThe present invention relates to a novel product and process for protection of animals or plants from arthropod infestation. According to the present invention there are provided arthropod esterase proteins and mimetopes thereof; arthropod nucleic acid molecules, including those that encode such proteins; antibodies raised against such esterase proteins (i.e., anti-arthropod esterase antibodies); and compounds that inhibit arthropod esterase activity (i.e, inhibitory compounds or inhibitors).
The present invention also includes methods to obtain such proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, mimetopes, nucleic acid molecules, antibodies, and/or inhibitory compounds, as well as use of such therapeutic compositions to protect animals from arthropod infestation.
Identification of an esterase of the present invention is unexpected, however, because the most similar nucleic acid sequence identified by previous investigators could not be used to identify an esterase of the present invention. In addition, identification of an esterase protein of the present invention is unexpected because a protein fraction from flea prepupal larvae that was obtained by monitoring for serine protease activity surprisingly also contained esterase proteins of the present invention.
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74.
The present invention also includes a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule encoding a protein comprising at least one of the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74; and particularly a nucleic acid molecule that hybridizes with a nucleic acid sequence that is a complement of a nucleic acid sequence encoding any of the amino acid sequences. A preferred nucleic acid molecule of the present invention includes a nucleic acid molecule comprising a nucleic acid sequence including SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, and allelic variants thereof.
The present invention also includes an isolated carboxylesterase nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID NO:53. SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 represent N-terminal amino acid sequences of carboxylesterases isolated from prepupal flea larvae, the production of which are described in the Examples of the present application.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes an isolated esterase protein that is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to (a) a nucleic acid molecule that includes at least one of the following nucleic acid sequences: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, and SEQ ID NO:71; and/or (b) a nucleic acid molecule encoding a protein including at least one of the following amino acid sequences: SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:74. One embodiment is a carboxylesterase protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule that encodes a protein comprising at least one of the following amino acid sequences: SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID NO:53. Preferred proteins of the present invention are isolated flea proteins including at least one of the following amino acid sequences: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74; also included are proteins encoded by allelic variants of nucleic acid molecules encoding proteins comprising any of the above-listed amino acid sequences.
The present invention also relates to mimetopes of arthropod esterase proteins as well as to isolated antibodies that selectively bind to arthropod esterase proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
The present invention also includes a formulation of flea carboxylesterase proteins, in which the proteins, when submitted to 14% Tris-glycine SDS-PAGE, comprise a fractionation profile as depicted in
Also included in the present invention is a formulation of flea carboxylesterase proteins, in which the proteins, when submitted to IEF-PAGE, comprise a fractionation profile as depicted in
Another embodiment of the present invention is an isolated flea protein or a formulation of flea proteins that hydrolyzes α-napthyl acetate to produce α-napthol, when the protein is incubated in the presence of α-napthyl acetate contained in 20 mM Tris at pH 8.0 for about 15 minutes at about 37° C.
Yet another embodiment of the present invention is an isolated flea protein or a formulation of flea proteins that hydrolyzes the methyl ester group of juvenile hormone to produce a juvenile hormone acid.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting flea carboxylesterase activity, the method comprising: (a) contacting an isolated flea carboxylesterase with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has carboxylesterase activity; and (b) determining if the putative inhibitory compound inhibits the activity. Also included in the present invention is a test kit to identify a compound capable of inhibiting flea carboxylesterase activity, the test kit comprising an isolated flea carboxylesterase protein having esterase activity and a means for determining the extent of inhibition of the activity in the presence of a putative inhibitory compound.
Yet another embodiment of the present invention is a therapeutic composition that is capable of reducing hematophagous ectoparasite infestation. Such a therapeutic composition includes at least one of the following protective compounds: an isolated hematophagous ectoparasite carboxylesterase protein or a mimetope thereof, an isolated carboxylesterase nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalides felis carboxylesterase gene, an isolated antibody that selectively binds to a hematophagous ectoparasite carboxylesterase protein, and an inhibitor of carboxylesterase activity identified by its ability to inhibit the activity of a flea carboxylesterase. A therapeutic composition of the present invention can also include an excipient, an adjuvant and/or a carrier. Preferred esterase nucleic acid molecule compounds of the present invention include naked nucleic acid vaccines, recombinant virus vaccines and recombinant cell vaccines. Also included in the present invention is a method to protect an animal from hematophagous ectoparasite infestation, comprising the step of administering to the animal a therapeutic composition of the present invention.
The present invention provides for isolated arthropod esterase proteins, isolated arthropod esterase nucleic acid molecules, antibodies directed against arthropod esterase proteins and other inhibitors of arthropod esterase activity. As used herein, the terms isolated arthropod esterase proteins and isolated arthropod esterase nucleic acid molecules refers to esterase proteins and esterase nucleic acid molecules derived from arthropods and, as such, can be obtained from their natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins, nucleic acid molecules, antibodies and inhibitors as therapeutic compositions to protect animals from hematophagous ectoparasite infestation as well as in other applications, such as those disclosed below.
Arthropod esterase proteins and nucleic acid molecules of the present invention have utility because they represent novel targets for anti-arthropod vaccines and drugs. The products and processes of the present invention are advantageous because they enable the inhibition of arthropod development, metamorphosis, feeding, digestion and reproduction processes that involve esterases. While not being bound by theory, it is believed that expression of arthropod esterase proteins are developmentally regulated, thereby suggesting that esterase proteins are involved in arthropod development and/or reproduction. The present invention is particularly advantageous because the proteins of the present invention were identified in larval fleas, thereby suggesting the importance of the proteins as developmental proteins.
One embodiment of the present invention is an esterase formulation that includes one or more esterase proteins capable of binding to diisopropylfluorophosphate (DFP). A preferred embodiment of an esterase formulation of the present invention comprises one or more arthropod esterase proteins that range in molecular weight from about 20 kilodaltons (kD) to about 200 kD, more preferably from about 40 kD to about 100 kD, and even more preferably from about 60 kD to about 75 kD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). An even more preferred formulation includes one or more flea esterase proteins having elution (or migration) patterns as shown in
Another embodiment of the present invention is a formulation comprising one or more hematophagous ectoparasite carboxylesterase (CE) proteins. The present invention includes the discovery that such a formulation has general CE activity. General CE activity can be identified using methods known to those of skill in the art and described in the Examples section herein. A suitable formulation of the present invention comprises one or more flea proteins capable of hydrolyzing α-napthyl acetate to produce α-napthyl when the proteins are incubated in the presence of α-napthyl acetate contained in 20 mm Tris at pH 8.0 for about 15 minutes at about 37° C. General CE activity can be identified following such incubation by detecting the production of from about 0.3 to about 2.5 absorbance units in the presence of Fast Blue when measured at 590 nm.
A preferred CE formulation of the present invention includes one or more flea CE proteins having acidic to neutral isoelectric points, or pI values. An isoelectric pH, or pI, value refers to the pH value at which a molecule has no net electric charge and fails to move in an electric field. A preferred formulation of the present invention includes one or more proteins having a pI value ranging from about pI 2 to about 10, more preferably from about pI 3 to about 8, and even more preferably from about pI 4.7 to about 5.2, as determined by IEF-PAGE.
An esterase formulation, including a CE formulation, of the present invention can be prepared by a method that includes the steps of: (a) preparing an extract by isolating flea tissue, homogenizing the tissue by sonication and clarifying the extract by centrifugation at a low speed spin, e.g., about 18,000 rpm for about 30 minutes; (b) recovering soluble proteins from said centrifuged extract and applying the proteins to a p-aminobenzamidine agarose bead column; (c) recovering unbound protein from the column and clarifying by filtration; (d) applying the clarified protein to a gel filtration column and eluting and collecting fractions with esterase activity; (e) dilayzing the eluate against 20 mM MES buffer, pH 6.0, containing 10 mM NaCl; (f) applying the dialysate to a cation exchange chromatography column, eluting protein bound to the column with a linear gradient of from about 10 mM NaCl to about 1 M NaCl in 20 mM MES buffer, pH 6, and collecting fractions having esterase activity; (g) adjusting the pH of the resulting fractions to pH 7 and applying the fractions to an anion exchange chromatography column; (h) eluting protein bound to the column with a linear gradient of from about 0 to about 1 M NaCl in 25 mM Tris buffer, pH 6.8 and collecting fractions having esterase activity, such activity elutes from the column at about 170 mM NaCl.
Tissue can be obtained from unfed fleas or from fleas that recently consumed a blood meal (i.e., blood-fed fleas). Such flea tissues are referred to herein as, respectively, unfed flea and fed flea tissue. Preferred flea tissue from which to obtain an esterase formulation of the present invention includes pre-pupal larval tissue, wandering flea larvae, 3rd instar tissue, fed adult tissue and unfed adult tissue.
In a preferred embodiment, a CE formulation of the present invention comprises a flea protein comprising amino acid sequence SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID NO:53.
Another embodiment of the present invention is a juvenile hormone esterase (JHE) formulation comprising one or more arthropod JHE proteins, the arthropod being of the order Hemiptera, Anoplura, Mallophaga, Diptera, Siphonaptera, Parasitiformes, Acariformes and Acarina. The present invention includes the discovery that such a formulation has JHE activity. JHE activity can be identified using methods known to those of skill in the art and described in the Examples section herein. A suitable formulation of the present invention comprises one or more arthropod proteins capable of hydrolyzing a methyl ester group of juvenile hormone to produce a juvenile hormone acid. Preferably, such a protein is capable of releasing of at least about 120 counts per minute when such a protein is incubated in the presence of 3H-juvenile hormone to create a reaction mixture, the reaction mixture is combined with isooctane, the aqueous phase is recovered and the amount of 3H-juvenile hormone present in that phase is determined. Such a protein is also preferably capable of causing release of methane thiol when such protein is incubated in the presence of methyl 1-heptylthioacetothioate (HEPTAT) using the method generally disclosed in McCutchen et al., Insect Biochem. Molec. Biol., Vol. 25, No. 1, pg 119-126, 1995, which is incorporated in its entirety by this reference.
In one embodiment, a juvenile hormone esterase formulation of the present invention comprises a protein comprising amino acid sequence SEQ ID NO:74.
According to the present invention, an arthropod that is not of the order lepidoptera includes an arthropod of the order Hemiptera, Anoplura, Mallophaga, Diptera, Siphonaptera, Parasitiformes, Acariformes and Acarina. Preferred arthropods include Hemiptera cimicidae, Hemiptera reduviidae, Anoplura pediculidae, Anoplura pthiridae, Diptera culicidae, Diptera simuliidae, Diptera psychodidae, Diptera ceratopogonidae, Diptera chaoboridae, Diptera tabanidae, Diptera rhagionidae, athericidae, Diptera chloropidae, Diptera muscidae, Diptera hippoboscidae, Diptera calliphoridae, Diptera sarcophagidae, Diptera oestridae, Diptera gastrophilidae, Diptera cuterebridae, Siphonaptera ceratophyllidae, Siphonaptera leptopsyllidae, Siphonaptera pulicidae, Siphonaptera tungidae, Parasitiformes dermanyssidae, Acariformes tetranychidae, Acariformes cheyletide, Acariformes demodicidae, Acariformes erythraeidae, Acariformes trombiculidae, Acariformes psoroptidae, Acariformes sarcoptidae, Acarina argasidae and Acarina ixodidae. Preferred Diptera muscidae include Musca, Hydrotaea, Stomoxys Haematobia. Preferred Siphonaptera include Ceratophyllidae nosopsyllus, Ceratophyllidae diamanus, Ceratophyllidae ceratophyllus, Leptopsyllidae leptopsylla, Pulicidae pulex, Pulicidae ctenocephalides, Pulicidae xenopsylla, Pulicidae echidnophaga and Tungidae tunga. Preferred Parasitiformes dermanyssidae include Ornithonyssus and Lilponyssoides. Preferred Acarina include Argasidae argas, Argasidae ornithodoros, Argasidae otobius, Ixodidae ixodes, Ixodidae hyalomma, Ixodidae nosomma, Ixodidae rhipicephalus, Ixodidae boophilus, Ixodidae dermacentor, Ixodidae haemaphysalus, Ixodidae amblyomma and Ixodidae anocentor.
One embodiment of a JHE formulation of the present invention is one or more arthropod JHE proteins that range in molecular weight from about 20 kD to about 200 kD, more preferably from about 40 kD to about 100 kD, and even more preferably from about 60 kD to about 75 kD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
A JHE formulation of the present invention can be prepared by a method that includes the steps of: (a) preparing soluble proteins from arthropod extracts as described above for CE purification and purifying such soluble proteins by gel filtration; (b) collecting fractions having JHE activity from the gel filtration step, loading the fractions onto a cation exchange column, eluting the cation exchange column with a linear gradient of from about 10 mM NaCl to about 1 M NaCl in 20 mM MES buffer, pH 6 and collecting fractions having JHE activity; (c) adjusting the pH of the collected fractions to about pH 7 are dialyzed against about 10 mM phosphate buffer (pH 7.2) containing about 10 mM NaCl; (d) applying the dialysate to a hydroxyapatite column, eluting protein bound to the column with a linear gradient of from about 10 mM phosphate buffer (pH 7.2) containing 10 mM NaCl to about 0.5 M phosphate buffer (pH 6.5) containing 10 mM NaCl and collecting fractions having JHE activity; (e) dialyzing the fractions against 20 mM Tris buffer (pH 8.0) containing 10 mM NaCl; (f) applying the dialysate an anion exchange chromatography column and eluting protein bound to the column with a linear gradient of from about 10 mM to about 1 M NaCl in 20 mM Tris buffer, pH 8 and collecting fractions containing SHE activity.
A JHE formulation of the present invention can be prepared by a method that includes the steps of: (a) preparing flea extracts as described herein in the Examples section and applying the extract to p-aminobenzamidine linked agarose beads and collecting protein not bound to the beads; (b) applying the unbound protein to a Superdex 200 HR gel filtration column and collecting fractions having JHE activity; (c) applying the fractions to an anion exchange chromatography column, eluting the anion exchange column with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH 6.8 and collecting fractions having JHE activity; (d) dialyzing the fractions overnight against about 1 L of 20 mM Tris buffer, pH 8.0, containing 10 mM NaCl; (e) applying the dialysate to a Poros 10 HQ anion exchange column, eluting the column with buffer containing about 120 mM NaCl and collecting fractions having JHE activity.
Suitable arthropods from which to isolate a JHE formulation of the present invention include, but are not limited to agricultural pests, stored product pests, forest pests, structural pests or animal health pests. Suitable agricultural pests of the present invention include, but are not limited to Colorado potato beetles, corn earworms, fleahoppers, weevils, pink boll worms, cotton aphids, beet armyworms, lygus bugs, hessian flies, sod webworms, whites grubs, diamond back moths, white flies, planthoppers, leafhoppers, mealy bugs, mormon crickets and mole crickets. Suitable stored product pests of the present invention include, but are not limited to dermestids, anobeids, saw toothed grain beetles, indian mealmoths, flour beetles, long-horn wood boring beetles and metallic wood boring beetles. Suitable forest pests of the present invention include, but are not limited to southern pine bark bettles, gypsy moths, elm beetles, ambrosia bettles, bag worms, tent worms and tussock moths. Suitable structural pests of the present invention include, but are not limited to, bess beetles, termites, fire ants, carpenter ants, wasps, hornets, cockroaches, silverfish, Musca domestics and Musca autumnalis. Suitable animal health pests of the present invention include, but are not limited to fleas, ticks, mosquitoes, black flies, lice, true bugs, sand flies, Psychodidae, tsetse flies, sheep blow flies, cattle grub, mites, horn flies, heel flies, deer flies, Culicoides and warble flies. Preferred arthropods from which to isolate a JHE formulation of the present invention include fleas, midges, mosquitoes, sand flies, black flies, horse flies, snipe flies, louse flies, horn flies, deer flies, tsetse flies, buffalo flies, blow flies, stable flies, meiosis-causing flies, biting gnats, lice, mites, bee, wasps, ants, true bugs and ticks, preferably fleas, ticks and blow flies, and more preferably fleas. Preferred fleas from which to isolate JHE proteins include Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla. More preferred fleas include Ctenocephalidesfelis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans, with C. felis being even more preferred.
Suitable tissue from which to isolate a JHE formulation of the present invention includes unfed fleas or fleas that recently consumed a blood meal (i.e., blood-fed fleas). Such flea tissues are referred to herein as, respectively, unfed flea and fed flea tissue. Preferred flea tissue from which to obtain a JHE formulation of the present invention includes pre-pupal larval tissue, 3rd instar tissue, fed or unfed adult tissue, with unfed adult gut tissue being more preferred than fed or unfed whole adult tissue. It is of note that a JHE formulation of the present invention obtained from pre-pupal larval tissue does not hydrolyze α-napthyl acetate.
Another embodiment of the present invention is an esterase formulation comprising a combination of one or more arthropod CE and JHE proteins of the present invention. Suitable arthropods from which to isolate a combined CE and JHE formulation include those arthropods described herein for the isolation of a JHE formulation of the present invention. Preferred arthropods from which to isolate a combined CE and JHE formulation include fleas, midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, buffalo flies, blow flies, stable flies, meiosis-causing flies, biting gnats, lice, bee, wasps, ants, true bugs and ticks, preferably fleas, ticks and blow flies, and more preferably fleas. Suitable flea tissue from which to isolate a combined CE and JHE formulation of the present invention includes 3rd instar tissue, fed or unfed adult tissue and unfed adult tissue, with unfed adult gut tissue being more preferred than fed or unfed whole adult tissue.
In one embodiment, a formulation of the present invention comprises an esterase having both CE and JHE activity. Preferably, a formulation of the present invention that comprises an esterase having both CE and JHE activity comprises a flea protein comprising amino acid sequence SEQ ID NO:8 and/or SEQ ID NO:37.
Another embodiment of the present invention is an isolated protein comprising an arthropod esterase protein. It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, a protein refers to one or more proteins or at least one protein. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology or can be produced by chemical synthesis.
As used herein, an isolated arthropod esterase protein can be a full-length protein or any homolog of such a protein. An isolated protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein's ability to elicit an immune response against arthropod esterase proteins, to hydrolyze α-napthyl acetate, to hydrolyze the methyl ester group of juvenile hormone or bind to DFP. Esterase proteins of the present invention include CE and JHE proteins. As such, an esterase protein of the present invention can comprise a protein capable of hydrolyzing α-napthyl acetate, hydrolyzing the methyl ester group of juvenile hormone and/or binding to DFP. Examples of esterase homologs include esterase proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of eliciting an immune response against an arthropod esterase protein. That is, when the homolog is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural arthropod esterase protein. The ability of a protein to effect an immune response, can be measured using techniques known to those skilled in the art. Esterase protein homologs of the present invention also include esterase proteins that hydrolyze α-napthyl acetate and/or that hydrolyze the methyl ester group of juvenile hormone.
Arthropod esterase protein homologs can be the result of natural allelic variation or natural mutation. Esterase protein homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant nucleic acid techniques to effect random or targeted mutagenesis.
Isolated esterase proteins of the present invention have the further characteristic of being encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to a gene encoding a Ctenocephalides felis protein (i.e., a C. felis esterase gene). As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989; Sambrook et al., ibid., is incorporated by reference herein in its entirety. Stringent hybridization conditions typically permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction. Formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.
As used herein, a C. felis esterase gene includes all nucleic acid sequences related to a natural C. felis esterase gene such as regulatory regions that control production of the C. felis esterase protein encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. In one embodiment, a C. felis esterase gene of the present invention includes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74. Nucleic acid sequence SEQ ID NO:1 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nfE1401, the production of which is disclosed in the Examples. The complement of SEQ ID NO:1 (represented herein by SEQ ID NO:3) refers to the nucleic acid sequence of the strand complementary to the strand having SEQ ID NO:1, which can easily be determined by those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a complete double helix with) the strand for which the sequence is cited.
Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nfE2364, the production of which is disclosed in the Examples. The complement of SEQ ID NO:4 is represented herein by SEQ ID NO:6.
Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nfE3421, the production of which is disclosed in the Examples. The complement of SEQ ID NO:7 is represented herein by SEQ ID NO:9.
Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nfE4524, the production of which is disclosed in the Examples. The complement of SEQ ID NO:10 is represented herein by SEQ ID NO:12.
Nucleic acid sequence SEQ ID NO:13 represents the deduced sequence of the coding strand of an apparent coding region of a complementary DNA (cDNA) nucleic acid molecule denoted herein as nfE51982, the production of which is disclosed in the Examples. The complement of SEQ ID NO:13 is represented herein by SEQ ID NO:15.
Nucleic acid sequence SEQ ID NO:18 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE61792, the production of which is disclosed in the Examples. The complement of SEQ ID NO:18 is represented herein by SEQ ID NO:20.
Nucleic acid sequence SEQ ID NO:24 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE72836, the production of which is disclosed in the Examples. The complement of SEQ ID NO:24 is represented herein by SEQ ID NO:26.
Nucleic acid sequence SEQ ID NO:30 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE82801, the production of which is disclosed in the Examples. The complement of SEQ ID NO:30 is represented herein by SEQ ID NO:32.
Nucleic acid sequence SEQ ID NO:36 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE92007, the production of which is disclosed in the Examples. The complement of SEQ ID NO:36 is represented herein by SEQ ID NO:38.
Nucleic acid sequence SEQ ID NO:57 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE52144, the production of which is disclosed in the Examples. The complement of SEQ ID NO:57 is represented herein by SEQ ID NO:59.
Nucleic acid sequence SEQ ID NO:67 represents the deduced sequence of the coding strand of an apparent coding region of a cDNA nucleic acid molecule denoted herein as nfE101987, the production of which is disclosed in the Examples. The complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69.
It should be noted that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of the present invention and apparent amino acid sequences of esterase proteins of the present invention.
In another embodiment, a C. felis esterase gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74. An allelic variant of a C. felis esterase gene is a gene that occurs at essentially the same locus (or loci) in the genome as the gene including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5′ or 3′ untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given arthropod since the genome is diploid and/or among a group of two or more arthropods.
The minimal size of an esterase protein homolog of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. As such, the size of the nucleic acid molecule encoding such a protein homolog is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence. It should also be noted that the extent of homology required to form a stable hybrid can vary depending on whether the homologous sequences are interspersed throughout the nucleic acid molecules or are clustered (i.e., localized) in distinct regions on the nucleic acid molecules. The minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich. As such, the minimal size of a nucleic acid molecule used to encode an esterase protein homolog of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of an esterase protein homolog of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, multiple genes, or portions thereof. The preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
One embodiment of the present invention includes an arthropod esterase protein having CE enzyme activity. Such a CE protein preferably includes: a catalytic triad of serine—histidine—glutamic acid as well as the essential amino acids arginine and aspartic acid at positions similar to those described for juvenile hormone esterase, for example by Ward et al., 1992, Int J Biochem 24: 1933-1941; this reference is incorporated by reference herein in its entirety. Analysis of the apparent full-length protein sequences disclosed herein indicates that each of these amino acid sequences includes these amino acid motifs, as well as surrounding consensus sequences.
Suitable arthropods from which to isolate esterase proteins having general CE activity of the present invention (including isolation of the natural protein or production of the protein by recombinant or synthetic techniques) preferably include insects and acarines but not Culicidae, Drosophilidae, Calliphoridae, Sphingidae, Lymantriidae, Noctuidae, Fulgoroidae and Aphididae. Preferred arthropods from which to isolate CE proteins having general CE activity include fleas, ticks, black flies, lice, true bugs, sand flies, Psychodidae, tsetse flies, cattle grub, mites, horn flies, heel flies, deer flies, Culicoides and warble flies. Preferred arthropods from which to isolate an esterase proteins having general CE activity include fleas, midges, sand flies, black flies, horse flies, snipe flies, louse flies, horn flies, deer flies, tsetse flies, buffalo flies, blow flies, stable flies, meiosis-causing flies, biting gnats, lice, mites, bee, wasps, ants, true bugs and ticks, preferably fleas, ticks and blow flies, and more preferably fleas. Preferred fleas from which to isolate esterase proteins having general CE activity include Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla. More preferred fleas include Ctenocephalides felis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans, with C. felis being even more preferred.
A preferred arthropod esterase protein of the present invention is a compound that when administered to an animal in an effective manner, is capable of protecting that animal from hematophagous ectoparasite infestation. In accordance with the present invention, the ability of an esterase protein of the present invention to protect an animal from hematophagous ectoparasite infestation refers to the ability of that protein to, for example, treat, ameliorate and/or prevent infestation caused by hematophagous arthropods. In particular, the phrase “to protect an animal from hematophagous ectoparasite infestation” refers to reducing the potential for hematophagous ectoparasite population expansion on and around the animal (i.e., reducing the hematophagous ectoparasite burden). Preferably, the hematophagous ectoparasite population size is decreased, optimally to an extent that the animal is no longer bothered by hematophagous ectoparasites. A host animal, as used herein, is an animal from which hematophagous ectoparasites can feed by attaching to and feeding through the skin of the animal. Hematophagous ectoparasites, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal in order to feed. At any given time, a certain percentage of a hematophagous ectoparasite population can be on a host animal whereas the remainder can be in the environment of the animal. Such an environment can include not only adult hematophagous ectoparasites, but also hematophagous ectoparasite eggs and/or hematophagous ectoparasite larvae. The environment can be of any size such that hematophagous ectoparasites in the environment are able to jump onto and off of a host animal. For example, the environment of an animal can include plants, such as crops, from which hematophagous ectoparasites infest an animal. As such, it is desirable not only to reduce the hematophagous ectoparasite burden on an animal per se, but also to reduce the hematophagous ectoparasite burden in the environment of the animal. In one embodiment, an esterase protein of the present invention can elicit an immune response (including a humoral and/or cellular immune response) against a hematophagous ectoparasite.
Suitable hematophagous ectoparasites to target include any hematophagous ectoparasite that is essentially incapable of infesting an animal administered an esterase protein of the present invention. As such, a hematophagous ectoparasite to target includes any hernatophagous ectoparasite that produces a protein having one or more epitopes that can be targeted by a humoral and/or cellular immune response against an esterase protein of the present invention and/or that can be targeted by a compound that otherwise inhibits esterase activity (e.g., a compound that inhibits hydrolysis of α-napthyl acetate, hydrolysis of the methyl ester group of juvenile hormone, and/or binds to DFP), thereby resulting in the decreased ability of the hematophagous ectoparasite to infest an animal. Preferred hematophagous ectoparasite to target include ectoparasites disclosed herein as being useful in the production of esterase proteins of the present invention.
The present invention also includes mimetopes of esterase proteins of the present invention. As used herein, a mimetope of an esterase protein of the present invention refers to any compound that is able to mimic the activity of such a protein (e.g., ability to elicit an immune response against an arthropod esterase protein of the present invention and/or ability to inhibit esterase activity), often because the mimetope has a structure that mimics the esterase protein. It is to be noted, however, that the mimetope need not have a structure similar to an esterase protein as long as the mimetope functionally mimics the protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); synthetic or natural organic or inorganic molecules, including nucleic acids; and/or any other peptidomimetic compounds. Mimetopes of the present invention can be designed using computer-generated structures of esterase proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, (e.g., an esterase substrate, an esterase substrate analog, or an anti-esterase antibody). A preferred mimetope is a peptidomimetic compound that is structurally and/or functionally similar to an esterase protein of the present invention, particularly to the active site of the esterase protein.
The present invention also includes mimetopes of esterase proteins of the present invention. As used herein, a mimetope of an esterase protein of the present invention refers to any compound that is able to mimic the activity of such an esterase protein, often because the mimetope has a structure that mimics the esterase protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
One embodiment of an arthropod esterase protein of the present invention is a fusion protein that includes an arthropod esterase protein-containing domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against an esterase protein; and/or assist purification of an esterase protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the esterase-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of an esterase protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an esterase-containing domain. Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a “tag” domain (e.g., at least a portion of β-galactosidase, a strep tag peptide, other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, Fla.; and an S10 peptide. Examples of particularly preferred fusion proteins of the present invention include PHIS-PfE6540, PHIS-PfE7275, PHIS-PfE7570, PHIS-PfE8570 and PHIS-PfE9528, production of which are disclosed herein.
In another embodiment, an arthropod esterase protein of the present invention also includes at least one additional protein segment that is capable of protecting an animal from hematophagous ectoparasite infestations. Such a multivalent protective protein can be produced by culturing a cell transformed with a nucleic acid molecule comprising two or more nucleic acid domains joined together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent protective compound containing at least two protective compounds, or portions thereof, capable of protecting an animal from hematophagous ectoparasite infestation by, for example, targeting two different arthropod proteins.
Examples of multivalent protective compounds include, but are not limited to, an esterase protein of the present invention attached to one or more compounds protective against one or more arthropod compounds. Preferred second compounds are proteinaceous compounds that effect active immunization (e.g., antigen vaccines), passive immunization (e.g., antibodies), or that otherwise inhibit a arthropod activity that when inhibited can reduce hematophagous ectoparasite burden on and around an animal. Examples of second compounds include a compound that inhibits binding between an arthropod protein and its ligand (e.g., a compound that inhibits flea ATPase activity or a compound that inhibits binding of a peptide or steroid hormone to its receptor), a compound that inhibits hormone (including peptide or steroid hormone) synthesis, a compound that inhibits vitellogenesis (including production of vitellin and/or transport and maturation thereof into a major egg yolk protein), a compound that inhibits fat body function, a compound that inhibits muscle action, a compound that inhibits the nervous system, a compound that inhibits the immune system and/or a compound that inhibits hematophagous ectoparasite feeding. Examples of second compounds also include proteins obtained from different stages of hematophagous ectoparasite development. Particular examples of second compounds include, but are not limited to, serine proteases, cysteine proteases, aminopeptidases, serine protease inhibitor proteins, calreticulins, larval serum proteins and echdysone receptors, as well as antibodies to and inhibitors of such proteins. In one embodiment, an arthropod esterase protein of the present invention is attached to one or more additional compounds protective against hematophagous ectoparasite infestation. In another embodiment, one or more protective compounds, such as those listed above, can be included in a multivalent vaccine comprising an arthropod esterase protein of the present invention and one or more other protective molecules as separate compounds.
A preferred isolated protein of the present invention is a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecules nfE1401, nfE2364, nfE3421, nfE4524, nfE51982, nfE51515, nfE52144, nfE51650, nfE61488, nfE61792, nfE61650, nfE72836, nfE71788, nfE71710, nfE7650, nfE82801, nfE81785, nfE81710, nfE92007, nfE91584, nfE91540, nfE101987 and/or nfE101590. A further preferred isolated protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69 and/or SEQ ID NO:71.
Translation of SEQ ID NO:1 suggests that nucleic acid molecule nfE1401 encodes a non-full-length arthropod esterase protein of about 103 amino acids, referred to herein as PfE1103, represented by SEQ ID NO:2, assuming the first codon spans from nucleotide 92 through nucleotide 94 of SEQ ID NO:1.
Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acid sequence of PfE1103) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:2, showed the most homology, i.e., about 33% identity, between SEQ ID NO:2 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:4 suggests that nucleic acid molecule nfE2364 encodes a non-full-length arthropod esterase protein of about 121 amino acids, referred to herein as PfE2121, represented by SEQ ID NO:5, assuming the first codon spans from nucleotide 2 through nucleotide 4 of SEQ ID NO:4.
Comparison of amino acid sequence SEQ ID NO:5 (i.e., the amino acid sequence of PfE2121) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:5, showed the most homology, i.e., about 38% identity, between SEQ ID NO:5 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:7 suggests that nucleic acid molecule nfE3421 encodes a non-full-length arthropod esterase protein of about 103 amino acids, referred to herein as PfE3103, represented by SEQ ID NO:8, assuming the first codon spans from nucleotide 113 through nucleotide 115 of SEQ ID NO:7.
Comparison of amino acid sequence SEQ ID NO:8 (i.e., the amino acid sequence of PfE3103) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:8, showed the most homology, i.e., about 39% identity, between SEQ ID NO:8 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:10 suggests that nucleic acid molecule nfE4524 encodes a non-full-length arthropod esterase protein of about 137 amino acids, referred to herein as PfF4137, represented by SEQ ID NO:11, assuming the first codon spans from nucleotide 113 through nucleotide 115 of SEQ ID NO:10.
Comparison of amino acid sequence SEQ ID NO:11 (i.e., the amino acid sequence of PfE4137) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:11, showed the most homology, i.e., about 30% identity, between SEQ ID NO:11 and Leptinotarsa decemlineata acetylcholinesterase.
Translation of SEQ ID NO:57 suggests that nucleic acid molecule nfE52144 encodes a full-length arthropod esterase protein of about 550 amino acids, referred to herein as PfE5550, represented by SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 32 of SEQ ID NO:57 and the termination (stop) codon spans from nucleotide 1680 through nucleotide 1682 of SEQ ID NO:57. The complement of SEQ ID NO:57 is represented herein by SEQ ID NO:59. The coding region encoding PfE5550 is represented by the nucleic acid molecule nfE51650, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:60 and a complementary strand with nucleic acid sequence SEQ ID NO:61. The deduced amino acid sequence of PfE5550 (i.e., SEQ ID NO:58) predicts that PfE5550 has an estimated molecular weight of about 61.8 kD and an estimated pI of about 5.5.
Comparison of amino acid sequence SEQ ID NO:58 (i.e., the amino acid sequence of PfE5550) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:58 showed the most homology, i.e., about 36% identity between SEQ ID NO:58 and Drosophila melanogaster alpha esterase protein.
Translation of SEQ ID NO:18 suggests that nucleic acid molecule nfE61792 encodes a full-length arthropod esterase protein of about 550 amino acids, referred to herein as PfE6550, represented by SEQ ID NO:19, assuming an open reading frame having an initiation codon spanning from nucleotide 49 through nucleotide 51 of SEQ ID NO:18 and a stop codon spanning from nucleotide 1699 through nucleotide 1701 of SEQ ID NO:18. The coding region encoding PfE6550, is represented by nucleic acid molecule nfE61650, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:21 and a complementary strand with nucleic acid sequence SEQ ID NO:22. The proposed mature protein, denoted herein as PfE6530, contains about 530 amino acids which is represented herein as SEQ ID NO:53. The nucleic acid molecule encoding PfE6530 is denoted herein as nfE61590 and has a coding strand having the nucleic acid sequence SEQ ID NO:23. The deduced amino acid sequence SEQ ID NO:19 suggests a protein having a molecular weight of about 61.8 kD and an estimated pI of about 5.5.
Comparison of amino acid sequence SEQ ID NO:19 (i.e., the amino acid sequence of PfE6550) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:19 showed the most homology, i.e., about 28% identity between SEQ ID NO:19 and Drosophila melanogaster alpha esterase protein.
Translation of SEQ ID NO:24 suggests that nucleic acid molecule nfE72836 encodes a full-length arthropod esterase protein of about 596 amino acids, referred to herein as PfE7596, represented by SEQ ID NO:25, assuming an open reading frame having an initiation codon spanning from nucleotide 99 through nucleotide 101 of SEQ ID NO:24 and a stop codon spanning from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:24. The coding region encoding PfE7596, is represented by nucleic acid molecule nfE71788, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:28 and a complementary strand with nucleic acid sequence SEQ ID NO:29. The proposed mature protein, denoted herein as PfE7570, contains about 570 amino acids which is represented herein as SEQ ID NO:54. The nucleic acid molecule encoding PfE7570 is denoted herein as nfE71710 and has a coding strand having the nucleic acid sequence SEQ ID NO:27. The deduced amino acid sequence SEQ ID NO:25 suggests a protein having a molecular weight of about 68.7 kD and an estimated pI of about 6.1.
Comparison of amino acid sequence SEQ ID NO:25 (i.e., the amino acid sequence of PfE7596) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:25 showed the most homology, i.e., about 27% identity between SEQ ID NO:25 and Drosophila melanogaster alpha esterase protein.
Translation of SEQ ID NO:30 suggests that nucleic acid molecule nfE82801 encodes a full-length arthropod esterase protein of about 595 amino acids, referred to herein as PfE8595, represented by SEQ ID NO:31, assuming an open reading frame having an initiation codon spanning from nucleotide 99 through nucleotide 101 of SEQ ID NO:30 and a stop codon spanning from nucleotide 1884 through nucleotide 1886 of SEQ ID NO:30. The coding region encoding PfE8595, is represented by nucleic acid molecule nfE81785, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:34 and a complementary strand with nucleic acid sequence SEQ ID NO:35. The proposed mature protein, denoted herein as PfE8570, contains about 570 amino acids which is represented herein as SEQ ID NO:55. The nucleic acid molecule encoding PfE8570 is denoted herein as nfE81710 and has a coding strand having the nucleic acid sequence SEQ ID NO:33. The deduced amino acid sequence SEQ ID NO:31 suggests a protein having a molecular weight of about 68.6 kD and an estimated pI of about 6.1.
Comparison of amino acid sequence SEQ ID NO:31 (i.e., the amino acid sequence of PfE8595) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:31 showed the most homology, i.e., about 28% identity between SEQ ID NO:31 and estalpha-2 esterase of Culex pipiens quinquefasciatus.
Translation of SEQ ID NO:36 suggests that nucleic acid molecule nfE92007 encodes a full-length arthropod esterase protein of about 528 amino acids, referred to herein as PfE9528, represented by SEQ ID NO:37, assuming an open reading frame having an initiation codon spanning from nucleotide 11 through nucleotide 13 of SEQ ID NO:36 and a stop codon spanning from nucleotide 1595 through nucleotide 1597 of SEQ ID NO:36. The coding region encoding PfE9528, is represented by nucleic acid molecule nfE91584, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:51 and a complementary strand with nucleic acid sequence SEQ ID NO:52. The deduced amino acid sequence SEQ ID NO:37 suggests a protein having a molecular weight of about 60 kD and an estimated pI of about 5.43.
Comparison of amino acid sequence SEQ ID NO:37 (i.e., the amino acid sequence of PfE9528) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:37 showed the most homology, i.e., about 37% identity between SEQ ID NO:37 and alpha esterase protein from Drosophila melanogaster.
Translation of SEQ ID NO:67 suggests that nucleic acid molecule nfE101987 encodes a full-length flea esterase protein of about 530 amino acids, referred to herein as PfE10530, having amino acid sequence SEQ ID NO:68, assuming an open reading frame in which the initiation codon spans from nucleotide 231 through nucleotide 233 of SEQ ID NO:67 and a stop codon spanning from nucleotide 1821 through nucleotide 1823 of SEQ ID NO:67. The complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69. The coding region encoding PfE10530, is represented by nucleic acid molecule nfE101590, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:70 and a complementary strand with nucleic acid sequence SEQ ID NO:71. The amino acid sequence of PfE10530 (i.e., SEQ ID NO:68) predicts that PfE10530 has an estimated molecular weight of about 59.5 kD and an estimated pI of about 5.5.
Comparison of amino acid sequence SEQ ID NO:68 (i.e., the amino acid sequence of PfE10530) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:68 showed the most homology, i.e., about 30% identity between SEQ ID NO:68 and Culex pipens esterase b1 precurser protein (SWISSPROT® # P16854).
More preferred arthropod esterase proteins of the present invention include proteins comprising amino acid sequences that are at least about 40%, preferably at least about 45%, more preferably at least about 50%, even more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and even more preferably at least about 95%, identical to amino acid sequence SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74.
More preferred arthropod esterase proteins of the present invention include proteins encoded by a nucleic acid molecule comprising at least a portion of nfE1401, nfE2364, nfE3421, nfE4524, nfE51982, nfE51515, nfE52144, nfE51650, nfE61488, nfE61792, nfE61650, nfE72836, nfE71788, nfE71710, nfE7650, nfE82801, nfE81785, nfE81710, nfE92007, nfE91584, nfE91540, nfE101987 and/or nfE101590, or of allelic variants of such nucleic acid molecules. More preferred is an esterase protein encoded by nfE1401, nfE2364, nfE3421, nfE4524, nfE51982, nfE51515, nfE52144, nfE51650, nfE61488, nfE61792, nfE61650, nfE72836, nfE71788, nfE71710, nfE7650, nfE82801, nfE81785, nfE81710, nfE92007, nfE91584, nfE91540, nfE101987 and/or nfE101590, or by an allelic variant of such nucleic acid molecules. Particularly preferred arthropod esterase proteins are PfE1103, PfE2121, PfE3103, PfE4137, PfE5505, PfE5550, PfE6550, PfE6530, PfE7596, PfE7570, PfE8595, PfE8570, PfE9528 and PfE10530.
In one embodiment, a preferred esterase protein of the present invention is encoded by at least a portion of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60 and/or SEQ ID NO:67, and, as such, has an amino acid sequence that includes at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58 and/or SEQ ID NO:68. Also preferred is a protein encoded by an allelic variant of a nucleic acid molecule comprising at least a portion of the above-listed nucleic acid sequences.
Particularly preferred esterase proteins of the present invention include SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74. (including, but not limited to, the proteins consisting of such sequences, fusion proteins and multivalent proteins) and proteins encoded by allelic variants of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60 and/or SEQ ID NO:67.
Another embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a C. felis esterase gene. The identifying characteristics of such a gene are heretofore described. A nucleic acid molecule of the present invention can include an isolated natural arthropod esterase gene or a homolog thereof, the latter of which is described in more detail below. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a nucleic acid molecule of the present invention is the minimal size that can form a stable hybrid with a C. felis esterase gene under stringent hybridization conditions.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified. An isolated arthropod esterase nucleic acid molecule of the present invention can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated esterase nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode an esterase protein of the present invention or to form stable hybrids under stringent conditions with natural gene isolates.
An arthropod esterase nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al, ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologs can be selected by hybridization with a C. felis esterase gene or by screening for the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of an arthropod esterase protein, hydrolyze α-napthyl acetate, hydrolyze the methyl ester group of juvenile hormone and/or bind to DFP).
An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one arthropod esterase protein of the present invention, examples of such proteins being disclosed herein. Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding an arthropod esterase protein.
A preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of protecting that animal from infestation by a hematophagous ectoparasite. As will be disclosed in more detail below, such a nucleic acid molecule can be, or can encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode a protective esterase protein (e.g., an esterase protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection (i.e, as a naked nucleic acid) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.
One embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE1401 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:1 and/or SEQ ID NO:3.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE2364 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:4 and/or SEQ ID NO:6.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE3421 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:7 and/or SEQ ID NO:9.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE4524 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:10 and/or SEQ ID NO:12.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE52144 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:57 and/or SEQ ID NO:59.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE61792 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:18 and/or SEQ ID NO:20.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE72836 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:24 and/or SEQ ID NO:26.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE82801 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:30 and/or SEQ ID NO:32.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE92007 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:36 and/or SEQ ID NO:38.
Another embodiment of the present invention is an esterase nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfE101987 and preferably with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:67 and/or SEQ ID NO:69.
Comparison of nucleic acid sequence SEQ ID NO:1 (i.e., the nucleic acid sequence of nfE1401) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:1 showed no identifiable identity with any sequence reported in GENBANK®.
Comparison of nucleic acid sequence SEQ ID NO:4 (i.e., the coding strand of nucleic acid sequence of nfE2364) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:4 showed the most homolog, i.e., about 43% identity, between SEQ ID NO:4 and a H. virescens juvenile hormone esterase gene.
Comparison of nucleic acid sequence SEQ ID NO:7 (i.e., the coding strand of nucleic acid sequence of nfE3421) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:7 showed the most homolog, i.e., about 53% identity, between SEQ ID NO:7 and a Torpedo marmorata acetylcholinesterase gene.
Comparison of nucleic acid sequence SEQ ID NO:10 (i.e., the coding strand of nucleic acid sequence of nfE4524) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:10 showed the most homolog, i.e., about 47% identity, between SEQ ID NO: 10 and an Anas platyrhyncos thioesterase B gene.
Comparison of nucleic acid sequence SEQ ID NO:57 (i.e., the coding strand of nucleic acid sequence of nfE52144) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:57 showed the most homolog, i.e., about 41% identity, between SEQ ID NO:57 and a esterase mRNA from Myzus persicae.
Comparison of nucleic acid sequence SEQ ID NO:18 (i.e., the coding strand of nucleic acid sequence of nfE61792) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:18 showed the most homolog, i.e., about 41% identity, between SEQ ID NO:18 and a esterase gene from Myzus persicae.
Comparison of nucleic acid sequence SEQ ID NO:24 (i.e., the coding strand of nucleic acid sequence of nfE72836) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:24 showed the most homolog, i.e., about 48% identity, between SEQ ID NO:24 and an Anas platychyncos thioesterase B gene.
Comparison of nucleic acid sequence SEQ ID NO:30 (i.e., the coding strand of nucleic acid sequence of nfE82801) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:30 showed the most homolog, i.e., about 46% identity, between SEQ ID NO:30 and a Mus musculus carboxyl ester lipase gene.
Comparison of nucleic acid sequence SEQ ID NO:36 (i.e., the coding strand of nucleic acid sequence of nfE92007) with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:36 showed the most homolog, i.e., about 47% identity, between SEQ ID NO:36 and a hamster mRNA for CE precursor gene.
Comparison of nucleic acid sequence SEQ ID NO:67 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:67 showed the most homology, i.e., about 48% identity, between SEQ ID NO:67 and a Lucilia cuprina alpha esterase gene (genembl # U56636) gene.
Preferred arthropod esterase nucleic acid molecules include nucleic acid molecules having a nucleic acid sequence that is at least about 55%, preferably at least about 60%, more preferably at least about 65%, more preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 90%, and even more preferably at least about 95% identical to nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74.
Another preferred nucleic acid molecule of the present invention includes at least a portion of nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, that is capable of hybridizing to a C. felis esterase gene of the present invention, as well as allelic variants thereof. A more preferred nucleic acid molecule includes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a protein comprising amino acid sequence SEQ ID NO:74, as well as allelic variants thereof. Such nucleic acid molecules can include nucleotides in addition to those included in the SEQ ID NOs, such as, but not limited to, a full-length gene, a full-length coding region, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule encoding a multivalent protective compound. Particularly preferred nucleic acid molecules include nfE1401, nfE2364, nfE3421, nfE4524, nfE51982, nfE51515, nfE52144, nfE51650, nfE61488, nfE61792, nfE61650, nfE72836, nfE71788, nfE71710, nfE7650, nfE82801, nfE81785, nfE81710, nfE92007, nfE91584, nfE91540, nfE11987 and nfE11590.
The present invention also includes a nucleic acid molecule encoding a protein having at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
Knowing the nucleic acid sequences of certain arthropod esterase nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain esterase nucleic acid molecules from other arthropods. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecule include flea pre-pupal, 3rd instar or adult cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources to screen or from which to amplify nucleic acid molecules include flea pre-pupal, 3rd instar or adult cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising arthropod esterase genes or other arthropod esterase nucleic acid molecules. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, primers to produce nucleic acid molecules or therapeutic reagents to inhibit esterase protein production or activity (e.g., as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents). The present invention also includes the use of such oligonucleotides to protect animals from disease using one or more of such technologies. Appropriate oligonucleotide-containing therapeutic compositions can be administered to an animal using techniques known to those skilled in the art.
One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulation of arthropod esterase nucleic acid molecules of the present invention.
One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells and more preferably in the cell types disclosed herein.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with arthropods, such as, C. felis.
Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein. Preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include nfE1401, nfE2364, nfE3421, nfE4524, nfE51982, nfE51515, nfE52144, nfE51650, nfE61488, nfE61792, nfE61650, nfE72836, nfE71788, nfE71710, nfE7650, nfE82801, nfE81785, nfE81710, nfE92007, nfE91584, nfE91540, nfE101987 and/or nfE101590. Particularly preferred recombinant molecules of the present invention include pCro-nfE61488, pTrc-nfE7650, pTrc-nfE71710, pTrc-nfE81710, pTrc-nfE51650, pTrc-nfE91540, pFB-nfE61679, pVL-nfE71802, pVL-fE81792 and pVL-nfE91600, the production of which are described in the Examples section.
Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed arthropod protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments, as well as natural signal sequences. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a cell include arthropod esterase nucleic acid molecules disclosed herein. Particularly preferred nucleic acid molecules with which to transform a cell include nfE1401, nfE2364, nfE3421, nfE4524, nfE51982, nfE51515, nfE52144, nfE51650, nfE61488, nfE61792, nfE61650, nfE72836, nfE71788, nfE71710, nfE7650, nfE82801, nfE81785, nfE81710, nfE92007, nfE91584, nfE91540, nfE101987 and/or nfE101590.
Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines). Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing arthropod esterase proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, other insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells. Particularly preferred host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 X3987 and SR-11 X4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK31 cells and/or HeLa cells. In one embodiment, the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
A recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed, examples of which are disclosed herein. Particularly preferred recombinant molecules include pCro-nfE61488, pTrc-nfE7650, pTrc-nfE71710, pTrc-nfE81710, pTrc-nfE51650, pTrc-nfE91540, pFB-nfE61679, pVL-nfE71802, pVL-fE81792 and pVL-nfE91600.
A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transform cells are disclosed herein. Particularly preferred recombinant cells include E. coli:pCro-nfE61488, E. coli:pTrc-nfE71710, E coli:pTrc-nfE7650, E. coli:pTrc-nfE81710, E. coli:pTrc-nfE51650, E. coli:pTrc-nfE91540, S. frugiperda:pVL-nfE71802, S. frugiperda:pVL-nfE81792, S. frugiperda:pVL-nfE91600 and S. frugiperda:pVL-nfE61679. Details regarding the production of these recombinant cells are disclosed herein.
Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including arthropod esterase nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other protective compounds, as disclosed herein (e.g., to produce multivalent vaccines).
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
Isolated esterase proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce an arthropod esterase protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Examples of suitable conditions are included in the Examples section.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane. The phrase “recovering the protein”, as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in “substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
The present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to an arthropod esterase protein of the present invention or a mimetope thereof (i.e., anti-arthropod esterase antibodies). As used herein, the term “selectively binds to” an esterase protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid. An anti-arthropod esterase antibody preferably selectively binds to an arthropod esterase protein in such a way as to reduce the activity of that protein.
Isolated antibodies of the present invention can include antibodies in a bodily fluid (such as, but not limited to, serum), or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to more than one epitope.
A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce arthropod esterase proteins of the present invention. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.
Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as therapeutic compounds to passively immunize an animal in order to protect the animal from arthropods susceptible to treatment by such antibodies and/or (b) as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present invention can be used to target cytotoxic agents to hematophagous ectoparasites such as those discloses herein, in order to directly kill such hematophagous ectoparasites. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled in the art. Suitable cytotoxic agents are known to those skilled in the art.
One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of protecting that animal from infestation by hematophagous ectoparasite. Therapeutic compositions of the present invention include at least one of the following protective compounds: an isolated hematophagous arthropod esterase protein (including a peptide); a mimetope of such a protein; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalides felis esterase gene; an isolated antibody that selectively binds to an hematophagous arthropod esterase protein; and inhibitors of hematophagous arthropod esterase activity (including esterase substrate analogs). As used herein, a protective compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent disease caused by an arthropod of the present invention. Preferred arthropods to target are heretofore disclosed. Examples of proteins, nucleic acid molecules, antibodies and inhibitors of the present invention are disclosed herein.
A preferred therapeutic composition of the present invention includes at least one of the following protective compounds: an isolated hematophagous ectoparasite carboxylesterase protein (including a peptide); a mimetope of such a protein; an isolated hematophagous ectoparasite carboxylesterase nucleic acid molecule that hybridizes under stringent hybridization conditions with a Ctenocephalides felis carboxylesterase gene; an isolated antibody that selectively binds to a hematophagous ectoparasite carboxylesterase protein; and an inhibitor of carboxylesterase activity identified by its ability to inhibit the activity of a flea carboxylesterase (including a substrate analog).
Suitable inhibitors of esterase activity are compounds that interact directly with an esterase protein's active site, thereby inhibiting that esterase's activity, usually by binding to or otherwise interacting with or otherwise modifying the esterase's active site. Esterase inhibitors can also interact with other regions of the esterase protein to inhibit esterase activity, for example, by allosteric interaction. Inhibitors of esterases are usually relatively small compounds and as such differ from anti-esterase antibodies. Preferably, an esterase inhibitor of the present invention is identified by its ability to bind to, or otherwise interact with, a flea esterase protein, thereby inhibiting the activity of the flea esterase.
Esterase inhibitors can be used directly as compounds in compositions of the present invention to treat animals as long as such compounds are not harmful to host animals being treated. Esterase inhibitors can also be used to identify preferred types of arthropod esterases to target using compositions of the present invention, for example by affinity chromatography. Preferred esterase inhibitors of the present invention include, but are not limited to, flea esterase substrate analogs, and other molecules that bind to a flea esterase (e.g., to an allosteric site) in such a manner that esterase activity of the flea esterase is inhibited; examples include, but are not limited to, juvenile hormone analogs and cholinesterase inhibitors as well as other neural transmission inhibitors. An esterase substrate analog refers to a compound that interacts with (e.g., binds to, associates with, modifies) the active site of an esterase protein. A preferred esterase substrate analog inhibits esterase activity. Esterase substrate analogs can be of any inorganic or organic composition, and, as such, can be, but are not limited to, peptides, nucleic acids, and peptidomimetic compounds. Esterase substrate analogs can be, but need not be, structurally similar to an esterase's natural substrate as long as they can interact with the active site of that esterase protein. Esterase substrate analogs can be designed using computer-generated structures of esterase proteins of the present invention or computer structures of esterases' natural substrates. Substrate analogs can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or other inorganic or organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, (e.g., a flea esterase). A preferred esterase substrate analog is a peptidomimetic compound (i.e., a compound that is structurally and/or functionally similar to a natural substrate of an esterase of the present invention, particularly to the region of the substrate that interacts with the esterase active site, but that inhibits esterase activity upon interacting with the esterase active site).
Esterase peptides, mimetopes and substrate analogs, as well as other protective compounds, can be used directly as compounds in compositions of the present invention to treat animals as long as such compounds are not harmful to the animals being treated.
The present invention also includes a therapeutic composition comprising at least one arthropod esterase-based compound of the present invention in combination with at least one additional compound protective against hematophagous ectoparasite infestation. Examples of such compounds are disclosed herein.
In one embodiment, a therapeutic composition of the present invention can be used to protect an animal from hematophagous ectoparasite infestation by administering such composition to a hematophagous ectoparasite, such as to a flea, in order to prevent infestation. Such administration could be oral, or by application to the environment (e.g., spraying). Examples of such compositions include, but are not limited to, transgenic vectors capable of producing at least one therapeutic composition of the present invention. In another embodiment, a hematophagous ectoparasite, such as a flea, can ingest therapeutic compositions, or products thereof, present in the blood of a host animal that has been administered a therapeutic composition of the present invention.
Compositions of the present invention can be administered to any animal susceptible to hematophagous ectoparasite infestation (i.e., a host animal), including warm-blooded animals. Preferred animals to treat include mammals and birds, with cats, dogs, humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits, raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail and turkeys as well as other furry animals, pets, zoo animals, work animals and/or food animals, being more preferred. Particularly preferred animals to protect are cats and dogs.
In accordance with the present invention, a host animal (i.e., an animal that is or is capable of being infested with a hematophagous ectoparasite) is treated by administering to the animal a therapeutic composition of the present invention in such a manner that the composition itself (e.g., an esterase inhibitor, an esterase synthesis suppressor (i.e., a compound that decreases the production of esterase in the hematophagous ectoparasite), an esterase mimetope, or an anti-esterase antibody) or a product generated by the animal in response to administration of the composition (e.g., antibodies produced in response to administration of an arthropod esterase protein or nucleic acid molecule, or conversion of an inactive inhibitor “prodrug” to an active esterase inhibitor) ultimately enters the hematophagous ectoparasite. A host animal is preferably treated in such a way that the compound or product thereof enters the blood stream of the animal. Hematophagous ectoparasites are then exposed to the composition or product when they feed from the animal. For example, flea esterase inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken up by feeding fleas. In another embodiment, when a host animal is administered an arthropod esterase protein or nucleic acid molecule, the treated animal mounts an immune response resulting in the production of antibodies against the esterase (i.e., anti-esterase antibodies) which circulate in the animal's blood stream and are taken up by hematophagous ectoparasites upon feeding. Blood taken up by hematophagous ectoparasites enters the hematophagous ectoparasites where compounds of the present invention, or products thereof, such as anti-esterase antibodies, esterase inhibitors, esterase mimetopes and/or esterase synthesis suppressors, interact with, and reduce esterase activity in the hematophagous ectoparasite.
The present invention also includes the ability to reduce larval hematophagous ectoparasite infestation in that when hematophagous ectoparasites feed from a host animal that has been administered a therapeutic composition of the present invention, at least a portion of compounds of the present invention, or products thereof in the blood taken up by the hematophagous ectoparasite are excreted by the hematophagous ectoparasite in feces, which is subsequently ingested by hematophagous ectoparasite larvae. In particular, it is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.
In accordance with the present invention, reducing esterase activity in a hematophagous ectoparasite can lead to a number of outcomes that reduce hematophagous ectoparasite burden on treated animals and their surrounding environments. Such outcomes include, but are not limited to, (a) reducing the viability of hematophagous ectoparasites that feed from the treated animal, (b) reducing the fecundity of female hematophagous ectoparasites that feed from the treated animal, (c) reducing the reproductive capacity of male hematophagous ectoparasites that feed from the treated animal, (d) reducing the viability of eggs laid by female hematophagous ectoparasites that feed from the treated animal, (e) altering the blood feeding behavior of hematophagous ectoparasites that feed from the treated animal (e.g., hematophagous ectoparasites take up less volume per feeding or feed less frequently), (f) reducing the viability of hematophagous ectoparasite larvae, for example due to the feeding of larvae from feces of hematophagous ectoparasites that feed from the treated animal and/or (g) altering the development of hematophagous ectoparasite larvae (e.g., by decreasing feeding behavior, inhibiting growth, inhibiting (e.g., slowing or blocking) molting, and/or otherwise inhibiting maturation to adults).
Therapeutic compositions of the present invention also include excipients in which protective compounds are formulated. An excipient can be any material that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, dog serum albumin, cat serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF); bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter's TITERMAX™ adjuvant (Vaxcel, Inc. Norcross, Ga.), Ribi adjuvants (Ribi Immunochem Research, Inc., Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein.
In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of an animal at a constant rate sufficient to attain therapeutic dose levels of the composition to protect an animal from hematophagous ectoparasite infestation. The therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months. A preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
Acceptable protocols to administer therapeutic compositions of the present invention in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram (μg) to about 10 milligrams (mg) of the therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 μg to about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal, intraocular and intramuscular routes.
According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein or protective RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNA drug) in the animal. Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid vaccine (e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or (b) administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
A naked nucleic acid vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A naked nucleic acid vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a bicistronic recombinant molecule having, for example one or more internal ribosome entry sites. Preferred naked nucleic acid vaccines include at least a portion of a viral genome (i.e., a viral vector). Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses, with those based on alphaviruses (such as Sindbis or Semliki virus), species-specific herpesviruses and species-specific poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production. Particularly preferred transcription control sequence include cytomegalovirus intermediate early (preferably in conjunction with Intron-A), Rous Sarcoma Virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of “strong” poly(A) sequences are also preferred.
Naked nucleic acid vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration being preferred. A preferred single dose of a naked nucleic acid vaccines ranges from about 1 nanogram (ng) to about 100 μg, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically. Naked DNA of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or a carrier (e.g., lipid-based vehicles).
A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging-deficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines is disclosed in PCT Publication No. WO 94/17813, by Xiong et al., published Aug. 18, 1994, which is incorporated by reference herein in its entirety.
When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from hematophagous ectoparasite infestation. For example, a recombinant virus vaccine comprising an arthropod CE nucleic acid molecule of the present invention is administered according to a protocol that results in the animal producing a sufficient immune response to protect itself from hematophagous ectoparasite infestation. A preferred single dose of a recombinant virus vaccine of the present invention is from about 1×104 to about 1×107 virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines, with subcutaneous, intramuscular, intranasal and oral administration routes being preferred.
A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 108 to about 1012 cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
The efficacy of a therapeutic composition of the present invention to protect an animal from hematophagous ectoparasite infestation can be tested in a variety of ways including, but not limited to, detection of anti-arthropod esterase antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with hematophagous ectoparasites to determine whether, for example, the feeding, fecundity or viability of hematophagous ectoparasites feeding from the treated animal is disrupted. Challenge studies can include attachment of chambers containing hematophagous ectoparasites onto the skin of the treated animal. In one embodiment, therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.
One preferred embodiment of the present invention is the use of arthropod protective compounds, such as proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds of the present invention, to protect an animal from hematophagous ectoparasite, and particularly flea, infestation. Preferred protective compounds of the present invention include, but are not limited to, C. felis esterase nucleic acid molecules, C. felis esterase proteins and mimetopes thereof, anti-C. felis esterase antibodies, and inhibitors of C. felis esterase activity. More preferred protective compounds of the present invention include, but are not limited to, CE or JHE formulations of the present invention, C. felis CE nucleic acid molecules, C. felis CE proteins and mimetopes thereof, anti-flea CE antibodies, anti-flea JHE antibodies, inhibitors of C. felis CE activity and inhibitors of flea JHE activity. Additional protection may be obtained by administering additional protective compounds, including other proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds, as disclosed herein.
One therapeutic composition of the present invention includes an inhibitor of arthropod esterase activity, i.e., a compound capable of substantially interfering with the function of an arthropod esterase susceptible to inhibition by an inhibitor of arthropod esterase activity. An inhibitor of esterase activity can be identified using arthropod esterase proteins of the present invention. One embodiment of the present invention is a method to identify a compound capable of inhibiting esterase activity of an arthropod. Such a method includes the steps of (a) contacting (e.g., combining, mixing) an isolated flea esterase protein, preferably a C. felis esterase protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has esterase activity, and (b) determining if the putative inhibitory compound inhibits the esterase activity. Putative inhibitory compounds to screen include small organic molecules, antibodies (including mimetopes thereof) and substrate analogs. Methods to determine esterase activity are known to those skilled in the art; see, for example, the Examples section of the present application.
The present invention also includes a test kit to identify a compound capable of inhibiting esterase activity of an arthropod. Such a test kit includes an isolated flea esterase protein, preferably a C. felis esterase protein, having esterase activity and a means for determining the extent of inhibition of esterase activity in the presence of (i.e., effected by) a putative inhibitory compound. Such compounds are also screened to identify those that are substantially not toxic in host animals.
Esterase inhibitors isolated by such a method, and/or test kit, can be used to inhibit any esterase that is susceptible to such an inhibitor. Preferred esterase proteins to inhibit are those produced by arthropods. A particularly preferred esterase inhibitor of the present invention is capable of protecting an animal from hematophagous ectoparasite infestation. Effective amounts and dosing regimens can be determined using techniques known to those skilled in the art.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
EXAMPLESIt is to be noted that the Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al., ibid., Borovsky, Arch Insect Biochem. and Phys., 7:187-210, 1988, and related references.
Example 1This example describes labeling of proteases and esterases with radiolabeled diisopropylfluorophosphate.
Tissue samples were isolated from unfed or bovine blood-fed 1st instar Ctenocephalides felis flea larvae; bovine blood-fed or cat blood-fed 3rd instar Ctenocephalides felis flea larvae; bovine blood-fed or cat blood-fed Ctenocephalides felis prepupal flea larvae; bovine blood-fed or cat blood-fed adult Ctenocephalides felis flea midgut tissue, and whole unfed, bovine blood-fed or cat blood-fed adult Ctenocephalides felis fleas. The 1st instar, 3rd instar, prepupal and adult midgut tissues were then homogenized by freeze-fracture and sonicated in a Tris buffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl2. The whole adult flea sample was then homogenized by freeze-fracture and ground with a microtube mortar and pestle. The extracts were centrifuged at about 14,000×g for 20 minutes (min.) and the soluble material recovered. The soluble material was then diluted to a final concentration of about 1 to about 1.2 tissue equivalents per microliter (μl) of Tris buffer. Each sample was labeled with [1,3-3H]-diisopropylfluorophosphate (3H-DFP) (available from DuPont-NEN, Wilmington, Del.) using the method generally described in Borovsky, ibid. About 20 tissue equivalents of each tissue sample were mixed with about 1 μCi of 3H-DFP and incubated for about 18 hours at 4° C. Proteins contained in each sample were then resolved using a 14% Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (available from Novex, San Diego, Calif.) under reducing conditions. The gel was soaked in Entensify (available from DuPont-NEN) according to manufacturers instructions, and exposed to X-ray film (available from Kodak X-0mat AR, Rochester, N.Y.) for about 3 days at −70° C.
Analysis of the resulting autoradiogram (shown in
This example describes the identification of general CE activity in flea tissue extracts.
Tissue samples and soluble extracts were prepared as described above in Example 1, except not labelled, from unfed (UF) and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed whole adult fleas, cat blood-fed adult (ACF) whole fleas, cat blood-fed adult fleas that have had their heads and midguts removed (referred to herein as fed adult partial fleas), unfed adult flea midguts and cat blood-fed adult flea midguts. About 5 tissue equivalents of each tissue were assayed for general CE activity using the following method. Tissue samples of about 5 μl were added to separate wells of flat-bottomed microtiter plate (available from Becton Dickinson, Lincoln Park, N.J.). A control well was prepared by adding about 5 μl of Tris buffer to an empty well of the plate. About 95 μl of 25 mM Tris-HCl (pH 8.0) was then added to each sample to increase the volume in each well to about 100 μl. About 100 μl of 0.25 mM α-napthyl acetate (available from Sigma-Aldrich, St. Louis, Mo.) dissolved in 25 mM Tris-HCl (pH 8.0) was then added to each well. The plate was then incubated for about 15 min. at 37° C. Following the incubation, about 40 μl of 0.3% Fast Blue salt BN (tetrazotized o-dianisidine; available from Signa-Aldrich) dissolved in 3.3% SDS in water was added to each well.
The microtiter plate was then analyzed using a Cambridge Technology, Inc. (Watertown, Pa.) model 7500 Microplate Reader set to 590 nm. The absorbance value for the control sample was subtracted from absorbance values of experimental samples, such that the background value was zero.
The results shown in
This example describes the determination of general CE activity using isoelectric focusing (JEF)-PAGE and non-reducing SDS-PAGE.
A. Non-Reducing SDS-PAGE.
Soluble extracts from unfed and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, bovine blood-fed adult (ABF) whole fleas and cat blood-fed adult whole fleas were prepared using the method described in Example 1. Each soluble extract sample was combined with SDS sample buffer (available from Novex) and proteins in the samples were resolved by gel electrophoresis using 14% Tris-glycine SDS electrophoresis gels (available from Novex). The gels were run at room temperature for about 1 hour at 200 volts. After electrophoresis, the gels were soaked for about for 30 minutes in 50 mM Tris, pH 8.0, containing 2.5% Triton X-100 to renature the proteins. The gels were then soaked in 50 mM Tris, pH 8.0, for about 5 minutes and then stained for about 5 min. in 50 milliliters (ml) of 25 mM Tris, pH 8.0, containing 50 mg Fast blue salt BN and 10 mg α-napthyl acetate (dissolved in 1 ml acetone). Once protein was detected on the stained gels, the gels were rinsed with water and photographed.
B. IEF-PAGE.
Soluble extracts from unfed and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed and cat blood-fed whole fleas, cat blood-fed adult partial fleas and cat blood-fed adult midguts were prepared as described above in Section A. The extracts were each combined with IEF sample buffer pH 3-7 (available from Novex) and loaded onto pH 3-7 IEF electrophoresis gels (available from Novex). The gels were electrophoresed at room temperature first for about 1 hour at about 100 volts, then for about 1 hour at about 200 volts, and then for about 30 min. at about 500 volts. Following electrophoresis, the gels were soaked in 25 mM Tris buffer, pH 8.0, for about 5 min. and then stained for about 1-5 min. in 50 ml of 25 mM Tris buffer, pH 8.0, containing 50 mg Fast blue salt BN and 10 mg α-napthyl acetate (dissolved in 1 ml acetone). Once protein was detected on the stained gels, the gels were rinsed with water and photographed.
C. Results.
The results from gel electrophoresis experiments described above in Sections A and B are shown in
This example describes the purification of CE protein from prepupal flea larvae.
About 15,000 bovine blood-fed prepupal flea larvae were collected and the larvae were homogenized in TBS by sonication in 50 ml Oak Ridge centrifuge tubes (available from Nalgene Co., Rochester, N.Y.) by sonicating 4 times 20 seconds each at a setting of 5 of a model W-380 Sonicator (available from Heat Systems-Ultrasonics, Inc.). The sonicates were clarified by centrifugation at 18,000 RPM for 30 minutes to produce an extract. Soluble protein in the extract was removed by aspiration and diluted to a volume of about 20 ml in TBS (equivalent to about 1 larva per μl TBS). The extract was then added to a column containing about 5 ml of p-aminobenzamidine linked to agarose beads (available from Sigma-Aldrich, St. Louis, Mo.) and incubated overnight at 4° C. The column was then washed with about 30 ml TBS to remove unbound protein. The collected unbound protein was then concentrated to a volume of about 20 ml using a Macrosep 10 centrifugal protein concentrator (Filtron Technology Corp., Northborough, Mass.) and filtered sequentially through a 1 μm syringe filter and then through a 0.2 μm syringe filter to clarify the sample for chromatography.
Aliquots of about 0.5 ml were loaded onto a 20 ml Superdex 200 HR gel filtration column (available from Pharmacia, Piscataway, N.J.) equilibrated in TBS, operated on a BioLogic liquid chromatography system (available from BioRad, Burlingame, Calif.). About 1 ml fractions were then collected. Repetitive runs were performed until about 30 ml of each fraction was collected. The fractions were analyzed for CE activity using the assay described above in Example 2. In preparation for cation exchange chromatography, fractions having CE activity (Ve=16-18 ml) were combined and dialyzed against about 2 liters of 20 mM MES buffer (2-(N-morpholino)ethanesulfonic acid), pH 6.0, containing 10 mM NaCl, for about 1.5 hours, and then against about 1 liter of the same buffer overnight at 4° C. Prior to loading onto the cation exchange chromatography column, the sample was again filtered through a 0.2 μm syringe filter to remove precipitated proteins. The sample was then applied to a Bio-Scale S2 cation exchange column (available from BioRad) at a rate of about 0.5 ml/min. The column was washed with MES buffer until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCl in 20 mM MES buffer, pH 6. Fractions were assayed for CE activity using the assay described above in Example 2. The results indicated that CE activity was not retained on the cation exchange column using the above conditions, and all of the activity was found in the flow-through fractions.
Fractions containing CE activity were pooled and adjusted to pH 7 using 0.5 M Tris, pH 8.0, in preparation for anion exchange chromatography. The pooled fractions were then loaded onto a 4.5 mm×50 mm Poros 10 HQ anion exchange chromatography column (available from PerSeptive Biosystems, Cambridge, Mass.) equilibrated in 25 mM Tris buffer, pH 6.8. The column was washed with the loading buffer, and bound proteins were eluted with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH 6.8. Fractions were tested for CE activity using the assay described above in Example 2. The results indicated that CE activity was eluted at about 170 mM NaCl. Fractions containing CE activity were pooled and diafiltered into TBS.
Example 5This example describes the determination of N-terminal amino acid sequences of carboxylesterases isolated from prepupal flea larvae.
A. Anion Exchange Chromatography Fractions.
Anion exchange chromatography fractions described above in Example 4 that contained proteins having CE activity were pooled, diafiltered into TBS buffer and concentrated 3-fold in a SPEEDVAC® Concentrator (available from Savant Instruments, Holbrook, N.Y.). Proteins in the concentrated samples were then resolved on a reducing, 10% SDS-PAGE Tris-glycine gel (available from Novex) for 1 hour at about 200 V. The proteins on the gel were then blotted onto a polyvinylidene difluoride (PVDF) membrane (available from Novex) for about 70 min in 10 mM CAPS buffer (3-[cyclohexylamino]-1-propanesulfonic acid; available from Sigma-Aldrich), pH 11, with 0.5 mM dithiothreitol (DTT). The membrane was then stained for 1 minute in 0.1% Coomassie Blue R-250 dissolved in 40% methanol and 1% acetic acid. The membrane was destained in 50% methanol for about 10 minutes, rinsed with MilliQ water and air dried. Three stained protein bands were identified having apparent molecular weights of about 64 kD, 65 kD, and 66 kD, respectively. The portion of the membrane containing each band was excised separately. Protein contained in each membrane segment was subjected to N-terminal amino acid sequencing using a 473A Protein Sequencer (available from Applied Biosystems, Foster City, Calif.) and using standard techniques.
The results indicated that the N-terminal amino acid sequence of the putative 64 kD protein was DPPTVTLPQGEL (denoted SEQ ID NO:39); the N-terminal amino acid sequence of the putative 65 kD protein was DPPTVTLPQGELVGKATNEnxk (denoted SEQ ID NO:40); and the N-terminal amino acid sequence of the putative 66 kD protein was DppTVTLPQGEL (denoted SEQ ID NO:41), in which the lower case letters designate uncertainties and “x” designates an undetermined residue.
B. Proteins Resolved by Native IEF-PAGE.
Proteins isolated by anion exchange chromatography as described above in Section A were further resolved by native IEF-PAGE. Proteins were loaded onto a pH 3-10 IEF gel (available from Novex) and separated in Novex's IEF buffers according to Novex's standard procedure (60 min at 100 V; then 60 min at 200 V; and then 30 min at 500 V). Following electrophoresis, part of the gel was stained for CE activity using the method described above in Example 2. The remaining portion of the gel was blotted onto PVDF membrane by reversing the orientation of the gel and membrane so that positively charged proteins migrated to the membrane, electrophoresing the protein for 60 min at 10 V, using 0.7% acetic acid as the transfer buffer. The membrane was stained as described above in Section A. After the membrane was dried, stained protein bands on the membrane were compared to bands on the gel tested for CE activity to identify corresponding bands. Protein bands on the membrane corresponding to proteins having CE activity were excised and submitted to N-terminal sequencing as described in Section A.
N-terminal amino acid sequence was obtained for protein contained in two bands having pI values of about pI 4.8 and about pI 4.9. N-terminal amino acid sequence of the pI 4.8 band was DPPTVTLPQGELVGKALSNen (denoted SEQ ID NO:42) and N-terminal amino acid sequence of the pI 4.9 band was DPPTVTLP (denoted SEQ ID NO:43). A comparison of the N-terminal amino acid sequences identified here and described in Section A indicates closely related proteins having a consensus sequence of DPPTVTLPQGELVGKALTNEnGk (denoted SEQ ID NO:44).
The amino acid sequences of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 are substantially contained within SEQ ID NO:5, SEQ ID NO:19 and SEQ ID NO:53, which are described below in Example 11.
Example 6This example describes partial purification of CE from 3rd instar flea larvae.
Using the extract preparation methods described in Example 1 without labelling, extracts were prepared from about 50,000 bovine blood-fed 3rd instar flea larvae. The extract was then further purified over a p-aminobenzamidine linked agarose bead column using the method also described in Example 1. Collected unbound protein was concentrated to about 70 ml using a 200 ml stirred cell fitted with a YM-10 membrane (available from Amicon, Beverly, Mass.). Seven ml (about 5,000 3rd instar flea larval equivalents) of the concentrated extract was used for the remainder of the purification scheme described in Example 4. Resulting fractions from the anion exchange chromatography column were tested for CE activity using the assay described above in Example 2.
The results indicated that CE activity was eluted in two overlapping peaks at about 120 mM and about 210 mM NaCl.
Example 7This example describes the identification of JHE activity in different flea tissues.
Tissue samples were prepared as described above in Example 1 from unfed and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed and cat blood-fed whole adult fleas, cat blood-fed adult partial fleas and cat blood-fed adult flea midguts. About 5 tissue equivalents of each tissue was assayed for JHE activity as follows.
Unlabeled juvenile hormone (JH; available from ICN Biomedicals, Inc., Aurora, Ohio) was diluted in hexane to concentration of about 0.025 M. Labeled 10-3H-juvenile hormone (3H-JH; available from Dupont-NEN) was diluted in hexane to concentration of about 80,000 cpm/μl. A JH substrate mixture was prepared by mixing about 20 μl of unlabeled JH with about 80 μl of 3H-JH (about 5 μCi) in a 4 ml screw cap vial. The substrate mixture was then covered with nitrogen (i.e., “blanketed”) and the solvent contained in the mixture was evaporated by heating the mixture at 35° C. When just dry, about 1 ml of absolute anhydrous ethanol (final concentration 5×10−4 M, or 6400 cpm/μl) was added to the vial. The substrate mixture was then stored at −20° C.
About 5 equivalents of each tissue (about 5 μl of protein) was added into the bottom of a small glass autosampler vial. About 95 μl of Tris-buffered saline (TBS) was added to each vial to bring the final volume in each vial to about 100 μl. Two control samples were also prepared by adding 100 μl TBS to two separate vials. About 1 μl of the substrate mixture described above was added to all of the vials including the control samples. The final JH concentration in each vial was about 5×10−6 M. The vials were then capped and spun in a microfuge to bring all of the liquid to the bottom of each vial. The vials were then transferred to a heat block and incubated at 35° C. for about 30 minutes. Following the incubation, enzyme activity was stopped by adding about 50 μl of methanol buffer (methanol:water:concentrated ammonium hydroxide at a 10:9:1 ratio, respectively) to each vial and removing the vials from the heat block.
To measure labeled juvenile hormone acid, about 250 μl isooctane was added to each vial. Each vial was vortexed for about 15 seconds or until an emulsion formed. Each vial was then centrifuged in a microfuge for about 1 minute to separate aqueous and organic phases. About 75 μl of the aqueous layer was removed from each vial and added to about 2 ml Eco-lume scintillation fluid (available from ICN). The amount of 3H-juvenile hormone acid contained in each vial was determined using a Beckman LS-1801 liquid scintillation counter (available from Beckman, Fullerton, Calif.).
The results shown in
This example describes the purification of JHE protein from cat blood-fed adult midguts.
About 23,000 cat blood-fed adult midguts were collected and prepared using the method described in Example 1. The extract was then added in 4 aliquots to columns containing about 3 to about 5 ml of p-aminobenzamidine linked agarose beads (available from Sigma-Aldrich), equilibrated in 50 mM Tris (pH 8.0), 100 mM CaCl2, 400 mM NaCl, and incubated overnight at 4° C. The columns were then washed with about 15 to about 125 ml of the equilibration Tris buffer to remove unbound protein. The collected unbound protein was pooled and then concentrated to a volume of about 5 ml using an Ultrafree-20 10 kD centrifugal concentrator (available from Millipore, Bedford, Mass.) and filtered sequentially through a 0.2 μm centrifugal ultrafiltration membrane (available from Lida, Kenosha, Wis.) to clarify the sample for chromatography.
Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR gel filtration column using the method described in Example 4. Repeated runs were performed until about 10 ml of each fraction was collected. The fractions were analyzed for JHE activity using the assay described in Example 7. In preparation for anion exchange chromatography, fractions having JHE activity (Ve=17-18 ml) were combined and dialyzed overnight against about 1 L of 20 mM Tris buffer, pH 8.0, containing 10 mM NaCl. The sample was then loaded onto a Poros 10 HQ anion exchange column using the method described in Example 4. Resulting fractions were tested for JHE activity as described in Example 7.
The results indicated that midgut JHE activity was eluted from the anion exchange column in a single peak at about 120 mM NaCl.
Example 9This example describes partial purification of JHE from prepupal flea larvae and 3rd instar larvae.
A. JHE Purification from Prepupal Tissue.
Using the extract preparation methods described in Example 1, gel filtration fractions were obtained using a Superdex 200 HR gel filtration column (available from Pharmacia) using the method described in Example 4, from about 15,000 bovine blood-fed prepupal flea larvae. The fractions were analyzed for JHE activity using the assay described above in Example 7. Those fractions containing protein having JHE activity (Ve=16-18 ml) were combined and dialyzed using the method described in Example 8.
The fractions were then further purified by passing the fractions over a Bio-Scale S2 cation exchange column (available from BioRad) at a rate of about 0.5 ml/min. The column was washed with MES until all unbound protein was eluted. Bound protein was then eluted with a linear gradient of 20 mM MES buffer, pH 6.0, containing 10 mM NaCl to 1 M NaCl. Resulting fractions were assayed for JHE activity using the method described in Example 7. The results indicated that proteins having JHE activity using prepupal tissue eluted from the column in about 200 to 300 mM NaCl.
The fractions containing JHE activity were combined and the pH adjusted to pH 7 using 0.5 M Tris buffer (pH 8.0). The fractions were then dialyzed twice against about 1 liter of 10 mM phosphate buffer (pH 7.2) containing 10 mM NaCl at 4° C. The resulting dialyzed fractions were then loaded onto a Bio-Scale CHT2-I Hydroxyapatite Column (available from BioRad) at a rate of about 0.5 ml/min. Unbound protein was washed from the column using the dialysis buffer. Bound protein was then eluted with a linear gradient of from 10 mM phosphate buffer, pH 7.2, containing 10 mM NaCl to 0.5 M phosphate buffer pH 6.5 containing 10 mM NaCl. One ml fractions were collected and each tested for JHE activity by the method described in Example 7.
The results indicated that JHE eluted in 2 overlapping peaks at about 100 mM and 150 mM phosphate. These two JHE activities were designated PP JHE I and PP JHE II, and were kept separate for the remainder of the purification. Both JHE samples were dialyzed overnight against 20 mM Tris buffer (pH 8.0) containing 10 mM NaCl. The two samples were then loaded, separately, onto a 4.5 mm×50 mm Poros 10 HQ anion exchange chromatography column (available from PerSeptive Biosystems) equilibrated with 20 mM Tris buffer, pH 8.0, containing 10 mM NaCl. Unbound proteins were washed from the column using the same buffer. Bound proteins were eluted with a linear gradient of from 10 mM to 1 M NaCl in 20 mM Tris buffer, pH 8.0. Resulting fractions were tested for JHE activity using the method described in Example 7.
The results indicated that in both samples, JHE activity was eluted from the column in a single peak at about 100 mM NaCl.
B. JHE Purification from 3rd Instar Tissue
Using the procedure described above in Section A, proteins having JHE activity were obtained using about 5,000 bovine blood-fed 3rd instar flea larvae. Following purification by cation exchange, proteins having JHE activity using 3rd instar tissue were found to elute in 2 peaks. The first peak having JHE activity was not retained on the column and also exhibited CE activity (referred to herein as CE/JHE fractions). The second peak having JHE activity eluted from the column in about 100-200 mM NaCl and did not contain CE activity.
The CE/JHE fractions were pooled and adjusted to about pH 7 using 0.5 M Tris, pH 8.0. The fractions were then loaded onto a 4.5 mm×50 mm Poros 10 HQ anion exchange chromatography column (available from PerSeptive Biosystems) and the column was equilibrated in 25 mM Tris buffer, pH 6.8. The column was washed with the same buffer and bound proteins were eluted with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH 6.8. Fractions were then tested for JHE activity using the method described in Example 7. JHE activity was eluted in two overlapping peaks at about 120 mM and 210 mM NaCl. The fraction containing JHE activity also contained CE activity when tested using the method described in Example 2.
Fractions from the cation exchange column containing only JHE activity were combined, diluted in 20 mM Tris buffer, pH 8.0 containing 10 mM NaCl, and concentrated to about 5 ml. The fractions were purified on a Poros 10 HQ anion exchange chromatography column as described immediately above. Fractions were then tested for JHE activity using the method described in Example 7. The JHE activity was eluted in a single peak at about 120 mM. The peak contained no detectable CE activity.
Example 10This example describes the purification of JHE protein from unfed adult midguts.
About 16,000 unfed adult midguts were collected in 20 mM Tris buffer (pH 7.7), containing 130 mM NaCl, 1 mM sodium EDTA, 1 mM PEFABLOC® (available from Boehringer Mannheim, Indianapolis, Ind.), 1 microgram/ml (μg/ml) leupeptin and 1 μg/ml pepstatin. The midguts were homogenized by freeze-fracture and sonication, and then centrifuged at about 14,000×g for 20 min. The soluble material from the centrifugation step was recovered. The soluble material was then concentrated to about 1 ml using an Ultrafree-20 10 kD centrifugal concentrator (available from Millipore) and filtered sequentially through a 0.2 μm centrifugal ultrafiltration membrane to clarify the sample for chromatography. Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR gel filtration column using the method described in Example 4. Repeated column runs were performed until about 2 ml of each fraction was collected. The fractions were analyzed for JHE activity using the assay described in Example 7. In preparation for cation exchange chromatography, fractions having JHE activity (Ve=15-17 ml) were combined and dialyzed overnight against about 1 L of 20 mM MES buffer, pH 6.0, containing 10 mM NaCl. The sample was then applied to a Bio-Scale S2 cation exchange column using the method described in Example 4. Fractions of eluate were assayed for JHE activity using the method described in Example 7.
The results indicate that JHE is present in unfed midguts in two forms, one that is not retained on the cation exchange column and one that is bound to the column under low salt conditions at about 100 mM NaCl. The form that was not retained under low salt conditions was shown to have general CE activity using the methods described in Example 2.
Example 11This example describes the identification of certain esterase nucleic acid molecules of the present invention.
Several flea esterase nucleic acid molecules, representing one or more partial flea esterase genes, were PCR amplified from a flea mixed instar cDNA library or a flea prepupal cDNA library. The flea mixed instar cDNA library was produced using unfed 1st instar, bovine blood-fed 1st instar, bovine blood-fed 2nd instar and bovine blood-fed 3rd instar flea larvae (this combination of tissues is referred to herein as mixed instar larval tissues for purposes of this example). The flea prepupal cDNA library was produced using prepupal flea larvae. For each library, total RNA was extracted from mixed instar or prepupal tissue, respectfully, using an acid-guanidinium-phenol-chloroform method similar to that described by Chomczynski et al., 1987, Anal. Biochem. 162, p. 156-159. Approximately 5,164 mixed instar larvae or 3,653 prepupal larvae were used in each RNA preparation. Poly A+ selected RNA was separated from each total RNA preparation by oligo-dT cellulose chromatography using POLY(A)QUICK® mRNA isolation kits (available from Stratagene Cloning Systems, La Jolla, Calif.), according to the method recommended by the manufacturer.
A mixed instar cDNA expression library and a prepupal cDNA expression library were constructed in lambda (λ) UNI-ZAP™XR vector (available from Stratagene Cloning Systems) using STRATAGENE® ZAP-cDNA® Synthesis Kit protocol. About 6.34 μg of mixed instar poly A+ RNA were used to produce the mixed instar library and about 6.72 μg of prepupal poly A+ RNA were used to produce the prepupal library. The resultant mixed instar library was amplified to a titer of about 2.17×1010 pfu/ml with about 97% recombinants. The resultant prepupal library was amplified to a titer of about 3.5×1010 pfu/ml with about 97% recombinants.
A pair of primers was used to amplify DNA from the cDNA libraries. A sense vector primer T-3X (corresponding to the vector in which nucleic acid molecules of the present invention had been ligated), having the nucleic acid sequence AATTAACCCT CACTAAAGGG (available from Gibco BRL, Gaithersburg, Md.; denoted SEQ ID NO:45), was used in combination with a degenerate primer, the design of which was based on a highly conserved esterase amino acid sequence (disclosed in Hanzlik et al., J. Biol. Chem. 264:12419-12423, 1989; I Y/H G G G F/L) located in a region downstream from the mature amino terminus in a number of known esterases. The degenerate primer, referred to herein as FCEF, is an anti-sense primer having the nucleic acid sequence ARDCCDCCDC CRTRDAT (R indicating an A or G; and D indicating an A, G or T; denoted SEQ ID NO:46). The resultant PCR products from the mixed instar cDNA library, obtained using standard PCR conditions (e.g., Sambrook et al., ibid.), were about 550 nucleotides. The resultant PCR products from the prepupal cDNA library were from about 500 nucleotides to about 860 nucleotides.
A. PCR Products.
PCR products were gel purified and cloned into the TA VECTOR™ (available from Invitrogen Corp., San Diego, Calif.). Approximately 8 clones were identified from the prepupal library and 6 clones were identified from the mixed instar library. These nucleic acid molecules were subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al., ibid.
1. Flea esterase clone 1 isolated from the mixed instar cDNA library was determined to comprise nucleic acid molecule nfE1401, the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:1. Translation of SEQ ID NO:1 suggests that nucleic acid molecule nfE1401 encodes a non-full-length flea esterase protein of about 103 amino acids, referred to herein as PfE1103, having amino acid sequence SEQ ID NO:2, assuming an initiation codon spanning from nucleotide 92 through nucleotide 94 of SEQ ID NO:1. The complement of SEQ ID NO:1 is represented herein by SEQ ID NO:3. Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acid sequence of PfE1103) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:2, showed the most homology, i.e., about 33% identity, between SEQ ID NO:2 and alpha esterase protein from Drosophila melanogaster.
2. Flea esterase clone 2 isolated from the mixed instar cDNA library was determined to comprise nucleic acid molecule nfE2364, the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:4. Translation of SEQ ID NO:4 suggests that nucleic acid molecule nfE2364 encodes a non-full-length flea esterase protein of about 121 amino acids, referred to herein as PfE2121, having amino acid sequence SEQ ID NO:5, assuming the first codon spans from nucleotide 2 through nucleotide 4 of SEQ ID NO:4. The complement of SEQ ID NO:4 is represented herein by SEQ ID NO:6. Comparison of nucleic acid sequence SEQ ID NO:4 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:4 showed the most homology, i.e., about 43% identity, between SEQ ID NO:4 and a H. virescens JHE gene. Comparison of amino acid sequence SEQ ID NO:5 (i.e., the amino acid sequence of PfE2121) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:5, showed the most homology, i.e., about 38% identity, between SEQ ID NO:5 and alpha esterase protein from Drosophila melanogaster.
3. Flea esterase clone 3 isolated from the prepupal cDNA library was determined to comprise nucleic acid molecule nfE3421, the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:7. Translation of SEQ ID NO:7 suggests that nucleic acid molecule nfE3421 encodes a non-full-length flea esterase protein of about 103 amino acids, referred to herein as PfE3103, having amino acid sequence SEQ ID NO:8, assuming an initiation codon spanning from nucleotide 113 through nucleotide 115 of SEQ ID NO:7. The complement of SEQ ID NO:7 is represented herein by SEQ ID NO:9. Comparison of nucleic acid sequence SEQ ID NO:7 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:7 showed the most homology, i.e., about 53% identity, between SEQ ID NO:7 and a Torpedo marmorata acetylcholinesterase gene. Comparison of amino acid sequence SEQ ID NO:8 (i.e., the amino acid sequence of PfE3103) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:8, showed the most homology, i.e., about 39% identity, between SEQ ID NO:5 and alpha esterase protein from Drosophila melanogaster.
4. Flea esterase clone 4 isolated from the prepupal cDNA library was determined to comprise nucleic acid molecule nfE4524, the nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:10. Translation of SEQ ID NO:10 suggests that nucleic acid molecule nfE4524 encodes a non-full-length flea esterase protein of about 137 amino acids, referred to herein as PfE4137, having amino acid sequence SEQ ID NO:11, assuming an initiation codon spanning from nucleotide 113 through nucleotide 115 of SEQ ID NO:10. The complement of SEQ ID NO:10 is represented herein by SEQ ID NO:12. Comparison of nucleic acid sequence SEQ ID NO:10 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:10 showed the most homology, i.e., about 47% identity, between SEQ ID NO:10 and an Anas platyrhyncos thioesterase B gene. Comparison of amino acid sequence SEQ ID NO:11(i.e., the amino acid sequence of PfE4137) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:11, showed the most homology, i.e., about 30% identity, between SEQ ID NO: 11 and Leptinotarsa decemlineata acetylcholinesterase.
B. cDNA Clones.
Certain amplified PCR fragments were used as probes to identify full-length flea esterase genes in the prepupal cDNA library.
1. Nucleic acid molecule nfE2364 was labeled with 32P and used as a probe to screen the mixed instar cDNA library described in Section A, using standard hybridization techniques. Two clones were isolated. A first clone included about a 2300-nucleotide insert, referred to herein as nfE52300. Nucleic acid sequence was obtained using standard techniques from nfE52300, to yield a flea esterase nucleic acid molecule named nfE51982 having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:13. Translation of SEQ ID NO:13 suggests that nucleic acid molecule nfE51982 encodes a non-full-length flea esterase protein of about 505 amino acids, referred to herein as PfE5505 having amino acid sequence SEQ ID NO:14, assuming the first codon spans from nucleotide 1 through nucleotide 3 of SEQ ID NO:13 and the stop codon spans from nucleotide 1518 through nucleotide 1520 of SEQ ID NO:13. The complement of SEQ ID NO:13 is represented herein by SEQ ID NO:15. The amino acid sequence of PfE5505 (i.e., SEQ ID NO:14) predicts that PfE5505 has an estimated molecular weight of about 56.8 kD and an estimated pI of about 5.5. The nucleic acid molecule representing the coding region for PfE5505 is referred to herein as nfE51515; the nucleic acid sequences of the coding strand and the complementary strand are represented by SEQ ID NO:16 and SEQ ID NO:17, respectively.
The nucleic acid sequence of nfE51982 was used to design primers to use in combination with a vector primer to PCR amplify the 5′ terminal fragment of the remainder of the flea esterase coding region from the flea mixed instar cDNA library. A pair of primers was used to amplify DNA from the cDNA library. A sense vector primer T3-X (corresponding to the vector in which nucleic acid molecules of the present invention had been ligated), having the nucleic acid sequence 5′ AATTAACCCT CACTAAAGGG 3′ (denoted SEQ ID NO:45), was used in combination with an anti-sense primer M6/M265′, having the nucleic acid sequence 5′ GTGCGTACAC GTTTACTACC 3′ (denoted SEQ ID NO:56). The resultant PCR product from the mixed instar cDNA library, obtained using standard PCR conditions (e.g., Sambrook et al., ibid.), were about 354 nucleotides.
The PCR product was subjected to DNA sequencing analysis, and a composite sequence representing a full-length flea esterase coding region was deduced. The nucleic acid sequence of the composite nucleic acid molecule, referred to herein as nfE52144 is denoted herein as SEQ ID NO:57+Translation of SEQ ID NO:57 suggests that nucleic acid molecule nfE52144 encodes a full-length flea esterase protein of about 550 amino acids, referred to herein as PfE5550, having amino acid sequence SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 32 of SEQ ID NO:57 and the stop codon spans from nucleotide 1680 through nucleotide 1682 of SEQ ID NO:57. The complement of SEQ ID NO:57 is represented herein by SEQ ID NO:59. The coding region encoding PfE5550 is represented by the nucleic acid molecule nfE51650, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:60 and a complementary strand with nucleic acid sequence SEQ ID NO:61. The amino acid sequence of PfE5550 (i.e., SEQ ID NO:58) predicts that PfE5550 has an estimated molecular weight of about 61.8 kD and an estimated pI of about 5.5.
Comparison of nucleic acid sequence SEQ ID NO:57 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:57 showed the most homology, i.e., about 41% identity, between SEQ ID NO:57 and a M. persicae esterase FE4 mRNA sequence. Comparison of amino acid sequence SEQ ID NO:58 (i.e., the amino acid sequence of PfE5550) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:58 showed the most homology, i.e., about 36% identity between SEQ ID NO:58 and Drosophila melanogster alpha esterase protein.
A second clone included about a 1900 nucleotide insert, referred to herein as nfE61900. Nucleic acid sequence was obtained using standard techniques from nfE61900, to yield a flea esterase nucleic acid molecule named nfE61792 having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:18. Translation of SEQ ID NO:18 suggests that nucleic acid molecule nfE61792 encodes a full-length flea esterase protein of about 550 amino acids, referred to herein as PfE6550, having amino acid sequence SEQ ID NO:19, assuming an open reading frame in which the initiation codon spans from nucleotide 49 through nucleotide 51 of SEQ ID NO:18 and a stop codon spanning from nucleotide 1699 through nucleotide 1701 of SEQ ID NO:18. The complement of SEQ ID NO: 8 is represented herein by SEQ ID NO:20. The coding region encoding PfE6550, is represented by nucleic acid molecule nfE61650, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:21 and a complementary strand with nucleic acid sequence SEQ ID NO:22. The proposed mature protein, denoted herein as PfE6530, contains about 530 amino acids which is represented herein as SEQ ID NO:53. The nucleic acid molecule encoding PfE6530 is denoted herein as nfE61590 and has a coding strand having the nucleic acid sequence SEQ ID NO:23. The amino acid sequence of PfE6550 (i.e., SEQ ID NO:19) predicts that PfE6550 has an estimated molecular weight of about 61.8 kD and an estimated pI of about 5.5.
Comparison of nucleic acid sequence SEQ ID NO:18 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:18 showed the most homology, i.e., about 41% identity, between SEQ ID NO:18 and a Myzus pericae esterase gene. Comparison of amino acid sequence SEQ ID NO:19 (i.e., the amino acid sequence of PfE6550) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:19 showed the most homology, i.e., about 28% identity between SEQ ID NO:19 and Drosophila melanogaster alpha esterase protein.
2. Nucleic acid molecule nfE4524 was labeled with 32P and used as a probe to screen the prepupal cDNA library described in Example 11, using standard hybridization techniques (e.g., Sambrook et al., ibid.). Two clones were isolated. A first clone included about a 3000 nucleotide insert, referred to herein as nfE73000. Nucleic acid sequence was obtained using standard techniques from nfE73000, to yield a flea esterase nucleic acid molecule named nfE72836 having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:24. Translation of SEQ ID NO:24 suggests that nucleic acid molecule nfE72836 encodes a full-length flea esterase protein of about 596 amino acids, referred to herein as PfE7596, having amino acid sequence SEQ ID NO:25, assuming an open reading frame in which the initiation codon spans from nucleotide 99 through nucleotide 101 of SEQ ID NO:24 and a stop codon spanning from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:25. The complement of SEQ ID NO:24 is represented herein by SEQ ID NO:26. The coding region encoding PfE7596, is represented by nucleic acid molecule nfE71788, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:28 and a complementary strand with nucleic acid sequence SEQ ID NO:29. The proposed mature protein, denoted herein as PfE7570, contains about 570 amino acids which is represented herein as SEQ ID NO:54. The nucleic acid molecule encoding PfE7570 is denoted herein as nfE71710 and has a coding strand having the nucleic acid sequence SEQ ID NO:27. The amino acid sequence of PfE7596 (i.e., SEQ ID NO:25) predicts that PfE7596 has an estimated molecular weight of about 68.7 kD and an estimated pI of about 6.1.
Comparison of nucleic acid sequence SEQ ID NO:24 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:24 showed the most homology, i.e., about 48% identity, between SEQ ID NO:24 and an Anas platychyncos thioesterase B gene. Comparison of amino acid sequence SEQ ID NO:25 (i.e., the amino acid sequence of PfE7596) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:25 showed the most homology, i.e., about 27% identity between SEQ ID NO:25 and Drosophila melanogaster alpha esterase protein.
A second clone included about a 3000 nucleotide insert, referred to herein as nfE83000. Nucleic acid sequence was obtained using standard techniques from nfE83000, to yield a flea esterase nucleic acid molecule named nfE82801 having a nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:30. Translation of SEQ ID NO:30 suggests that nucleic acid molecule nfE82801 encodes a full-length flea esterase protein of about 595 amino acids, referred to herein as PfE8595, having amino acid sequence SEQ ID NO:31, assuming an open reading frame in which the initiation codon spans from nucleotide 99 through nucleotide 101 of SEQ ID NO:30 and a stop codon spanning from nucleotide 1884 through nucleotide 1886 of SEQ ID NO:30. The complement of SEQ ID NO:30 is represented herein by SEQ ID NO:32. The coding region encoding PfE8595, is represented by nucleic acid molecule nfE81785, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:34 and a complementary strand with nucleic acid sequence SEQ ID NO:35. The proposed mature protein, denoted herein as PfE8570, contains about 570 amino acids which is represented herein as SEQ ID NO:55. The nucleic acid molecule encoding PfE8570 is denoted herein as nfE81710 and has a coding strand having the nucleic acid sequence SEQ ID NO:33. The amino acid sequence of PfE8595 (i.e., SEQ ID NO:31) predicts that PfE8595 has an estimated molecular weight of about 68.6 kD and an estimated pI of about 6.1.
Comparison of nucleic acid sequence SEQ ID NO:30 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:30 showed the most homology, i.e., about 46% identity, between SEQ ID NO:30 and a Mus musculus carboxyl ester lipase gene. Comparison of amino acid sequence SEQ ID NO:31 (i.e., the amino acid sequence of PfE8595) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:31 showed the most homology, i.e., about 28% identity between SEQ ID NO:31 and estalpha-2 esterase of Culex pipiens quinque fasciatus.
3. Nucleic acid molecule nfE3421 was labeled with 32P and used as a probe to screen the prepupal cDNA library using standard hybridization techniques (e.g., Sambrook et al., ibid.). Two clones were isolated. One clone included about a 1900 nucleotide insert, referred to herein as nfE91900. Nucleic acid sequence was obtained using standard techniques from nfE91900, to yield a flea esterase nucleic acid molecule named nfE92007 having nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:36. Translation of SEQ ID NO:36 suggests that nucleic acid molecule nfE92007 encodes a full-length flea esterase protein of about 528 amino acids, referred to herein as PfE9528, having amino acid sequence SEQ ID NO:37, assuming an open reading frame in which the initiation codon spans from nucleotide 11 through nucleotide 13 of SEQ ID NO:36 and a stop codon spanning from nucleotide 1595 through nucleotide 1597 of SEQ ID NO:36. The complement of SEQ ID NO:36 is represented herein by SEQ ID NO:38. The coding region encoding PfE9528, is represented by nucleic acid molecule nfE91584, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:51 and a complementary strand with nucleic acid sequence SEQ ID NO:52. The amino acid sequence of PfE9528 (i.e., SEQ ID NO:37) predicts that PfE9528 has an estimated molecular weight of about 60 kD and an estimated pI of about 5.43.
Comparison of nucleic acid sequence SEQ ID NO:36 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:36 showed the most homology, i.e., about 47% identity, between SEQ ID NO:36 and a hamster mRNA for carboxylesterase precursor gene. Comparison of amino acid sequence SEQ ID NO:37 (i.e., the amino acid sequence of PfE9528) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:37 showed the most homology, i.e., about 37% identity between SEQ ID NO:37 and alpha esterase protein from Drosophila melanogaster.
As is the case for any of the nucleic acid molecules described in this example, variations between sequences may be due to a number of factors, such as but not limited to, sequencing errors or allelic variation.
4. Nucleic acid molecule nfE1401 was labeled with 32P and used as a probe to screen the mixed instar cDNA library using standard hybridization techniques (e.g., Sambrook et al., ibid.). A clone was isolated that included about a 2000 nucleotide insert, referred to herein as nfE102000. Nucleic acid sequence was obtained using standard techniques from nfE102000, to yield a flea esterase nucleic acid molecule named nfE101987 having nucleic acid sequence of the coding strand which is denoted herein as SEQ ID NO:67. Translation of SEQ ID NO:67 suggests that nucleic acid molecule nfE101987 encodes a full-length flea esterase protein of about 530 amino acids, referred to herein as PfE10530, having amino acid sequence SEQ ID NO:68, assuming an open reading frame in which the initiation codon spans from nucleotide 231 through nucleotide 233 of SEQ ID NO:67 and a stop codon spanning from nucleotide 1821 through nucleotide 1823 of SEQ ID NO:67. The complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69. The coding region encoding PfE10530, is represented by nucleic acid molecule nfE101590, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:70 and a complementary strand with nucleic acid sequence SEQ ID NO:71. The amino acid sequence of PfE10530 (i.e., SEQ ID NO:68) predicts that PfE10530 has an estimated molecular weight of about 59.5 kD and an estimated pI of about 5.5.
Comparison of nucleic acid sequence SEQ ID NO:67 with nucleic acid sequences reported in GENBANK® indicates that SEQ ID NO:67 showed the most homology, i.e., about 48% identity, between SEQ ID NO:67 and a lucilia cuprina alpha esterase gene (genembl #U56636) gene. Comparison of amino acid sequence SEQ ID NO:68 (i.e., the amino acid sequence of PfE10530) with amino acid sequences reported in GENBANK® indicates that SEQ ID NO:68 showed the most homology, i.e., about 30% identity between SEQ ID NO:68 and Culex pipens esterase b1 precurser protein (SWISSPROT®# P16854).
As is the case for any of the nucleic acid molecules described in this example, variations between sequences may be due to a number of factors, such as but not limited to, sequencing errors or allelic variation.
Example 12This Example demonstrates the production of esterase proteins of the present invention in E. coli cells.
A. Flea esterase protein PHIS-PfE7570 and flea esterase protein PHIS-PfE8570 were produced in the following manner. A pair of primers was used to amplify DNA from flea esterase nucleic acid molecule nfE72836 or nfE82801 produced as described in Example 11. A sense primer containing an XhoI site (shown in bold) having the nucleic acid sequence 59 TGTGCTCGAG ATGGGATAAC CTAGATCAGC ATTTGTGC 3′ (denoted SEQ ID NO:47), was used in combination with an anti-sense primer containing a KpnI site (shown in bold) having the nucleic acid sequence 5′ TTAAGGTACC TCATCTAATA CTTCCTTCAT TACAG 3′ (denoted SEQ ID NO:48). A PCR product was derived from nfE72836, and is referred to herein as nfE71710, having nucleic acid sequence SEQ ID NO:27. The PCR product was digested with XhoI and KpnI restriction endonucleases, gel purified and subcloned into expression vector pTrcHisB (available from Invitrogen). The resultant recombinant molecule, referred to herein as pTrc-nfE71710, was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli:pTrc-nfE71710.
The PCR product derived from nfE82801 using the primers is referred to herein as nfE81710, having nucleic acid sequence SEQ ID NO:33. PCR product nfE81710 was digested with XhoI and KpnI restriction endonucleases, gel purified and subcloned into expression vector pTrcHisB. The resultant recombinant molecule, referred to herein as pTrc-nfE81710, was transformed into E. coli HB101 competent cells to form recombinant cell E. coli:pTrc-nfE81710.
The recombinant cells were cultured in enriched bacterial growth medium containing 0.1 mg/ml ampicillin and 0.1% glucose at about 32° C. When the cells reached an OD600 of about 0.4-0.5, expression of recombinant protein was induced by the addition of 0.5 mM isopropyl-B-D-thiogalactoside (IPTG), and the cells were cultured for about 2 hours at about 32° C. Immunoblot analysis of recombinant cell E. coli:pTrc-nfE71710 and E. coli:pTrc-nfE81710 lysates using a T7 tag monoclonal antibody (available from Novagen, Inc., Madison, Wis.) directed against the fusion portion of the recombinant PHIS-PfE7570 and PHIS-PfE8570 fusion proteins identified proteins of appropriate size, namely an about 65 kD protein for each fusion protein.
B. Flea esterase protein PHIS-PfE6540 was produced in the following manner.
A pair of primers was used to amplify DNA from flea esterase nucleic acid molecule nfE61792 produced as described in Example 11. A sense primer containing an XhoI site having the nucleic acid sequence 5′ AAACTCGAGT CCCCCGACTG TAACTTTGC 3′ (denoted SEQ ID NO:62; XhoI site shown in bold), was used in combination with an anti-sense primer containing a PstI site having the nucleic acid sequence 5′ TCATCTGCAG TTATTGACTG TGCAAAGTTT TTGTGG 3′ (denoted SEQ ID NO:63; PstI site shown in bold). A PCR product was derived from nfE61792, and is referred to herein as nfE61488, having nucleic acid sequence SEQ ID NO:76. The PCR product was digested with XhoI and PstI restriction endonucleases, gel purified and subcloned into expression vector lambdaPR/T2ori/S10HIS-RSET-A9, that had been digested with XhoI and PstI and dephosphorylated. The resultant recombinant molecule, referred to herein as pCro-nfE61488, was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli:pCro-nfE61488.
The recombinant cells were cultured using the method generally described in Section A of this example, except that the cells were grown under heat shift conditions rather than in the presence of IPTG. The cells were grown at 32° C. for about 2 hours, and then grown at 42° C. Immunoblot analysis of recombinant cell E. coli:pCro-nfE61488 lysate using a T7 tag monoclonal antibody directed against the fusion portion of the recombinant PHIS-PfE6540 fusion protein identified proteins of appropriate size, namely an about 60 kD protein for each fusion protein.
Expression of the recombinant PHIS-PfE6540 fusion protein was improved by transforming supercoiled plasmid pCro-nfE61488 DNA harvested from E. coli:pCro-nfE61488 cells into the BL-21 strain of E. coli (available from Novagen). The amount of expression PHIS-PfE6540 was confirmed by immunoblot using the method described immediately above.
E. coli cells expressing PHIS-PfE6540 protein were harvested from about 2 liters of media and suspended in about 140 ml of 50 mM Tris, pH 8.0, 50 mM NaCl, 0.1 mM phenylmethylsulfonylfluoride (PMSF) (Solubilization Buffer). The cells were broken by passage through a microfluidizer at 30 psi for 30 cycles. The sample was centrifuged at about 16,000×g for 30 min at 4° C. The supernatant S1) was recovered and the pellet was resuspended in about 80 ml of Solubilization Buffer and centrifuged at about 16,000×g for 30 min at 4° C. The supernatant (S2) was recovered and the pellet was resuspended in about 80 ml of Solubilization Buffer containing 0.1% Triton-X100 and centrifuged at about 16,000×g for 30 min at 4° C. The supernatant (S3) was recovered and the pellet was resuspended in about 140 mls 50 mM Tris, pH 8.0, 8 M Urea, 0.1 M PMSF and centrifuged at about 16,000×g. The supernatant (S4) was recovered and the pellet was resuspended in 40 mls 50 mM Tris, 8 M Urea, 0.1 M PMSF. Aliquots of each pellet and supernatant were analyzed by SDS-PAGE and immunoblot using the T7 tag monoclonal antibody described above. The results indicated that the PHIS PfE6540 protein was located in the final supernatant (S4). The PHIS-PfE6540 protein was loaded onto a 5.0 ml, Metal chelating HiTrap column charged with NiCl2 (obtained from Pharmacia Biotech Inc., Piscataway, N.J.), previously equilibrated with 50 mM Tris, 1 mM PMSF, 1 mM β-mercaptoethanol (βME), 8 M urea, pH 8.0 (Buffer A). The column was washed with 10 column volumes (CV) Buffer A and then with 10 cv with 50 mM Tris, 25 mM sodium acetate, 1 mM PMSF, 1 mM βME, 8 M urea, pH 6.0 (Buffer B) to remove loosely bound proteins. Bound PHIS-PfE6540 protein was eluted with 10 cv of 50 mM Tris, 25 mM sodium acetate, 1 mM PMSF, 1 mM βME, 8 M urea, pH 4.0 (Buffer C). Column fractions were analyzed for the presence of PHIS-PfE6540 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the majority of the PHIS-PfE6540 protein was eluted by Buffer C. The fractions containing the PHIS-PfE6540 protein were combined and loaded onto a 5 ml SP-Sepharose HiTrap column (obtained from Pharmacia Biotech Inc.) previously equilibrated with 50 mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM βME, 8 M Urea, pH 4.5 (SP-Sepharose Buffer). The column was washed with SP-Sepharose Buffer until most of the unbound protein was removed. Bound protein was eluted with an increasing salt gradient to 1 M NaCl over 100 ml (20 cv) in SP-sepharose buffer. Column fractions were analyzed for the presence of PHIS-PfE6540 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the PHIS-PfE6540 protein was eluted at about 0.75 M NaCl.
The purified PHIS-PfE61488 protein was used to produce an anti-M6 polyclonal antiserum as follows. Rabbits were immunized with PHIS-PfE61488 protein diluted to a concentration of about 0.1 mg/ml in PBS. One milliliter of the dilution was mixed 1:1 mix with Complete Freunds Adjuvant. In the primary immunization, about 500 μl of the 1:1 mmix was injected subcutaneously into 5 different sites (0.1 ml/site) and 500 μl was injected intradermally into 5 different sites (0.1 ml/site) on the rabbit. Booster shots were administered to the rabbit intramuscularly in 4 sites using 250 μl/site of a 1:1 mix of PHIS-PfE61488 protein with Incomplete Freunds Adjuvant. The booster shots were administered at days 14 and 35. Serum samples were obtained prior to immunization (pre-bleed), and at day 14 after primary immunization and day 14 after the first and second boost.
C. Flea esterase protein PHIS-PfE9528 was produced in the following manner.
A pair of primers was used to amplify DNA from flea esterase nucleic acid molecule nfE92007 produced as described in Example 11. A sense primer containing an BamHI site having the nucleic acid sequence 5′-TTC CGG ATC CGG CTG ATC TAC AAG TGA CTT TG-3′ (denoted SEQ ID NO:64; BamHI site shown in bold), was used in combination with an anti-sense primer containing a XhoI site having the nucleic acid sequence 5′ TGG TAC TCG AGT CAT AAA AAT TTA TTC CAA AAT C 3′ (denoted SEQ ID NO:65; XhoI site shown in bold). A PCR product was derived from nfE92007, and is referred to herein as nfE91540, having nucleic acid sequence SEQ ID NO:51. The PCR product was digested with BamI and XhoI restriction endonucleases, gel purified and subcloned into expression vector pTrcHisB (available from Invitrogen). The resultant recombinant molecule, referred to herein as pTrc-nfE91540, was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli:pTrc-nfE9540.
The recombinant cells were cultured using the method described in Section A of this example. Immunoblot analysis of recombinant cell E. coli:pTrc-nfE91540 lysate using a T7 tag monoclonal antibody directed against the fusion portion of the recombinant PHIS-PfE9528 fusion protein identified proteins of appropriate size, namely an about 59 kD protein for each fusion protein.
Expression of the recombinant PHIS-PfE9528 fusion protein was improved by transforming supercoiled plasmid pTrc-nfE91584 DNA harvested from E. coli:pTrc-nfE91540 cells into the BL-21 strain of E. coli. The amount of expression PHIS-PfE9528 was confirmed by immunoblot using the method described immediately above.
Two liters of media from cultures of E. coli cells expressing PHIS PfE9528 protein were harvested and S4 supernatant was prepared using the method described above in section B. PHIS PfE9528 protein contained in the S4 supernatant was loaded onto a 5.0 ml, Metal chelating HiTrap column, charged with NiCl2 (available from Pharmacia Biotech Inc., Piscataway, N.J.), previously equilibrated with 50 mM Tris, 1 mM PMSF, 1 mM βME, 8 M urea, pH 8.0 (Buffer A). The column was washed with 5 cv of Buffer A until all unbound protein was removed. Bound protein was eluted with a linear gradient from Buffer A to 50 mM Tris, 1 mM PMSF, 1 mM βME, 8 M urea, 1 M NaCl, pH 4.0. Column fractions were analyzed for the presence of PHIS pfE9528 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the majority of the PHIS pfE9528 protein was eluted at about 250 mM NaCl. The fractions containing the PHIS pfE9528 protein were combined and loaded onto a C4-reversed phase column (obtained from Vydak, Hesperia, Calif.), previously equilibrated with 0.05% trifluoroacetic acid (TFA). The column was washed with 0.05% TFA until all unbound protein was removed. Bound proteins were eluted with a linear gradient from 0.05% TFA to 0.05% TFA in acetonitrile. Column fractions were analyzed for the presence of PHIS pfE9528 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the PHIS pfE9528 protein was eluted at about 40% acetonitrile. The fractions containing the PHIS pfE9528 protein were combined and loaded onto a 5 ml Q-Sepharose HiTrap column previously equilibrated with 50 mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM βME, 8 M Urea, pH 8.5 (Q-Sepharose Buffer). The column was washed with Q-Sepharose Buffer until all unbound protein was removed. Bound protein was eluted with an increasing salt gradient to 1 M NaCl over 100 ml (20 cv) in Q-sepharose buffer. Column fractions were analyzed for the presence of PHIS-PfE9528 protein by immunoblot using the T7 tag monoclonal antibody as described above. The results indicated that the PHIS-PfE9528 protein was eluted at about 0.3 M NaCl.
The purified PHIS-PfE9528 protein was used to produce an anti-P1 polyclonal antiserum as follows. Rabbits were immunized with PHIS-PfE9528 protein diluted to a concentration of about 0.1 mg/ml in PBS. One milliliter of the dilution was mixed 1:1 mix with Complete Freunds Adjuvant. In the primary immunization, about 500 μl of the 1:1 mix was injected subcutaneously into 5 different sites (0.1 ml/site) and 500 μl was injected intradermally into 5 different sites (0.1 ml/site) on the rabbit. Booster shots were administered to the rabbit intramuscularly in 4 sites using 250 μl/site of a 1:1 mix of PHIS-PfE9528 protein with Incomplete Freunds Adjuvant. The booster shots were administered at days 14 and 35. Serum samples were obtained prior to immunization (pre-bleed), and at day 14 after primary immunization and day 14 after the first and second boost.
D. Flea esterase protein PHIS-PfE7275 was produced in the following manner.
A 650-bp fragment was produced by digesting nfE72836 DNA with the restriction enzymes BamHI and BgIII. The BamHI and BgIII fragment derived from nfE72836 is referred to herein as nfE7650, having nucleic acid sequence SEQ ID NO:72 and amino acid SEQ ID NO:73. The fragment was purified using a QIAQUICK™ Kit (available from Qiagen, Santa Clarita, Calif.), according to methods provided by the manufacturer. The purified fragment was subcloned into expression vector pTrcH is C which had been digested with BamHI and BgIII. The resultant recombinant molecule, referred to herein as pTrc-nfE7650 was transformed into E. coli DH-5a competent cells (available from Gibco BRL) to form recombinant cell E. coli:pTrc-nfE7650.
The recombinant cells were cultured using the method described above in section A. Immunoblot analysis of recombinant cell E. coli:pTrc-nfE7650 lysate using a T7 tag monoclonal antibody directed against the fusion portion of the recombinant PHIS-PfE7275 fusion protein identified proteins of appropriate size, namely an about 35 kD protein for each fusion protein.
Expression of the recombinant fusion protein was improved by transforming supercoiled plasmid pTrc-nfE7650 DNA harvested from E. coli:pTrc-nfE7650 cells into the BL-21 strain of E. coli. The amount of expression E. coli:pTrc-nfE7650 was confirmed by immunoblot using the method described immediately above.
Example 13This Example demonstrates the production of esterase proteins of the present invention in eukaryotic cells.
A. Recombinant molecule pBv-nfE71788, containing a flea esterase nucleic acid molecule spanning nucleotides from about 99 through about 1886 of SEQ ID NO:24, and pBv-nfE81785, containing a flea esterase nucleic acid molecule spanning nucleotides from about 99 through about 1883 of SEQ ID NO:30 each, operatively linked to baculovirus polyhedron transcription control sequences were produced in the following manner. In order to subclone a flea esterase nucleic acid molecule into baculovirus expression vectors, flea esterase nucleic acid molecule-containing fragments were separately PCR amplified from nfE72836 or nfE82801 DNA. A PCR fragment of 1858 nucleotides, named nfE71858, was amplified from nfE72836 using a sense primer E1113 FWD having the nucleic acid sequence 5′-AAAACTGCAG TATAAATATG TTACCTCACA GTAGTG-3′ (SEQ ID NO:49; PstI site shown in bold) and an antisense primer E 1113/2212 REV having the nucleic acid sequence 5′-TGCTCTAGAT TATCTAATAC TTCCTTCATT ACAG (SEQ ID NO:50; XbaI site shown in bold). A PCR fragment of 1858 nucleotides, named nfE81858, was amplified from nfE82801 using a sense primer E2212 FWD having the nucleic acid sequence 5′-AAAACTGCAG TATAAATATG TTACCTCACA GTGCATTAG-3′ (SEQ ID NO:66; PstI site shown in bold), and the antisense primer E 1113/2212 REV. The N-terminal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
In order to produce a baculovirus recombinant molecule capable of directing the production of PfE7596, the about 1,802 base pair PCR product (referred to as Bv-nfE71802) was digested with PstI and XbaI and subcloned into unique PstI and XbaI sites of pVL1392 baculovirus shuttle plasmid (available from Pharmingen, San Diego, Calif.) to produce the recombinant molecule referred to herein as pVL-nfE71802.
In order to produce a baculovirus recombinant molecule capable of directing the production of PfE8595, the about 1,792 base pair PCR product (referred to as Bv-nfE81792) was digested with PstI and XbaI and subcloned into PstI and XbaI digested to produce the recombinant molecule referred to herein as pVL-nfE81792.
The resultant recombinant molecules, pVL-nfE71802 and pVL-nfE8792, were verified for proper insert orientation by restriction mapping. Such a recombinant molecule can be co-transfected with a linear Baculogold baculovirus DNA (available from Pharmingen) into S. frugiperda Sf9 cells (available from Invitrogen) to form the recombinant cells denoted S. frugiperda:pVL-nfE71802 and S. frugiperda:pVL-fE81792. S. frugiperda:pVL-nfE71802 can be cultured in order to produce a flea esterase protein PfE7596. S. frugiperda:pVL-nfE81792 can be cultured in order to produce a flea esterase protein PfE8595.
B. Recombinant molecule pBv-PfE9528, containing a flea esterase nucleic acid molecule spanning nucleotides from 14 through 1595 of SEQ ID NO:36, operatively linked to baculovirus polyhedron transcription control sequences were produced in the following manner. In order to subclone a flea esterase nucleic acid molecule into baculovirus expression vectors, a flea esterase nucleic acid molecule-containing fragment was PCR amplified from nfE92007 DNA. A PCR fragment of about 1600 nucleotides, named nfE91600, was amplified from nfE92007 using a sense primer P121B1 Sense having the nucleic acid sequence 5′-CGC GGA TCC GCT GAT CTA CAA GTG ACT TTG C-3′ (SEQ ID NO:75; BamHI site shown in bold) and an antisense primer P121B1 Anti having the nucleic acid sequence 5′-CCG AGC GGC CGC ATA AAA ATT TAT TCC AAA ATC TAA GTC G-3′ (SEQ ID NO:76; NotI site shown in bold). The N-terminal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
In order to produce a baculovirus recombinant molecule capable of directing the production of PfE9528, the about 1,600 base pair PCR product (referred to as Bv-nfE91600) was digested with BamHI and NotI and subcloned into unique BamHI and NotI sites of pVL1393 baculovirus shuttle plasmid (available from Pharmingen, San Diego, Calif.) to produce the recombinant molecule referred to herein as pVL-nfE91600.
The resultant recombinant molecule, pVL-nfE91600, was verified for proper insert orientation by restriction mapping. Such a recombinant molecule can be co-transfected with a linear Baculogold baculovirus DNA into S. frugiperda Sf9 cells to form the recombinant cells denoted S. frugiperda:pVL-nfE91600. S. frugiperda:pVL-nfE91600 can be cultured in order to produce a flea esterase protein PfE9528.
An immunoblot of supernatant from cultures of S. frugiperda:pVL-nfE91600 cells producing the flea esterase protein PfE9528 was performed using the anti-P1 polyclonal antiserum described in detail in Example 12. Blots were incubated using serum samples from the pre-bleed or from serum collected 14 days after the first boost of the rabbit. Analysis of the supernatant from cultures of S. frugiperda:pVL-nfE91600 cells identified an about 66 kD protein.
C. Recombinant molecule pBv-PfE6530, containing a flea esterase nucleic acid molecule spanning nucleotides from 50 through 1701 of SEQ ID NO:18, operatively linked to baculovirus polyhedron transcription control sequences were produced in the following manner. In order to subclone a flea esterase nucleic acid molecule into baculovirus expression vectors, a flea esterase nucleic acid molecule-containing fragment was PCR amplified from nfE61792 DNA. A PCR fragment of about 1679 nucleotides, named nfE101679, was amplified from nfE61792 using a sense primer M6M32 Sense having the nucleic acid sequence 5′-GCG AGG CCT TAT AAA TAT GTC TCG TGT TAT TTT TTT AAG TTG-3′ (SEQ ID NO:75; StuI site shown in bold) and an antisense primer M6M32 Anti having the nucleic acid sequence 5′-GCA CTG CAG TTA TTG ACT GTG CAA AGT TTT TGT GG-3′ (SEQ ID NO:76; PstI site shown in bold). The N-terminal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
In order to produce a baculovirus recombinant molecule capable of directing the production of PfE6530, the about 1,679 base pair PCR product (referred to as Bv-nfE61679) was digested with StuI and PstI and subcloned into unique StuI and PstI sites of FAST BAC™ baculovirus shuttle plasmid (obtained from Gibco-BRL) to produce the recombinant molecule referred to herein as pFB-nfE61679.
The resultant recombinant molecule, pFB-nfE61679, was verified for proper insert orientation by restriction mapping. Such a recombinant molecule can be transformed into E. coli strain DH10 (obtained from Gibco-BRL) according to the manufacturer's instructions. The pFB-nfE61679 isolated from the transformed DH10 cells can then be co-transfected with a linear Baculogold baculovirus DNA into S. frugiperda Sf9 cells to form the recombinant cells denoted S. frugiperda:pFB-nfE61679.
S. frugiperda:pFB-nfE61679 can be cultured in order to produce a flea esterase protein PfE6530.
An immunoblot of supernatant from cultures of S. frugiperda:pFB-nfE61679 cells producing the flea esterase FfE6530 was performed using the anti-M6 polyclonal antiserum described in detail in Example 12. Blots were incubated using serum samples from the pre-bleed or from serum collected 14 days after the first boost of the rabbit. Analysis of the supernatant from cultures of S. frugiperda:pFB-nfE61679 cells identified an about 66 kD protein.
N-terminal amino acid sequence was obtained using standard methods for the about 66 kD protein identified using the anti-M6 polyclonal antiserum. The N-terminal amino acid sequence was determined to be identical to the N-terminal amino acid sequence of SEQ ID NO:44.
Example 14This example describes the purification of carboxylesterase protein from fed flea midguts.
About 43,000 cat blood-fed adult flea midguts were collected and prepared as previously described in Example 1. The extract was then added in 2 aliquots to columns containing about 1 to about 2 ml of p-aminobenzamidine linked agarose beads (available from Sigma-Aldrich), equilibrated in 50 mM Tris (pH 8.0), 400 mM NaCl, and incubated overnight at 4° C. The columns were then drained to remove unbound protein and the two aliquots of unbound protein were combined. The collected unbound protein was then concentrated and diafiltered into a total volume of about 16 ml of 25 mM Tris (pH 8), 10 mM NaCl using an Ultrafree-20 10 kD centrifugal concentrator (available from Millipore, Bedford, Mass.).
Aliquots of about 8 ml were loaded onto an Uno Q6 anion exchange column (available from Bio-Rad, Hercules, Calif.) equilibrated in 25 mM Tris (pH 8), 10 mM NaCl, operated on a BioLogic liquid chromatography system (available from Bio-Rad). The column was washed with 25 mM Tris (pH 8), 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCl in 25 mM Tris, pH 8. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was eluted at about 220 mM NaCl.
Fractions containing CE activity were pooled and diafiltered into a total volume of about 3 ml of 20 mM MES buffer (2-(N-morpholino)ethanesulfonic acid), pH 6.0, containing 10 mM NaCl, in preparation for cation exchange chromatography. The sample was then applied to an Uno S1 cation exchange column (available from Bio-Rad) equilibrated in MES buffer. The column was washed with MES buffer until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCl in 20 mM MES buffer, pH 6. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was not retained on the cation exchange column using the above conditions, and all of the activity was found in the flow-through fractions.
Fractions containing CE activity were pooled and diafiltered into a total volume of about 3 ml of 25 mM Tris (pH 8), 10 mM NaCl, in preparation for an additional anion exchange chromatography step. The sample was then applied to a Bio-Scale Q2 anion exchange column (available from Bio-Rad). The column was washed with 25 mM Tris (pH 8), 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCl in 25 mM Tris, pH 8. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was eluted at about 130 mM NaCl.
A fraction containing CE activity was diluted into a total volume of about 4 ml of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl, in preparation for hydroxyapatite chromatography. The sample was then applied to a Bio-Scale CHT2-I column (available from Bio-Rad) at a flow rate of about 0.5 ml/min. The column was washed with 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl to 0.5 M 10 mM phosphate buffer, pH 6.5 containing 10 mM NaCl. Fractions were assayed for CE activity using the assay described previously. The results indicated that CE activity was eluted at about 200 mM phosphate.
Example 15This example describes the purification of a carboxylesterase protein from wandering flea larvae.
About 120,000 bovine blood-fed adult wandering flea larvae were homogenized in 3 batches of about 40,000 wandering larvae in each batch, in Tris buffered saline (TBS), pH 8.0 as previously described, except that about 1.2 mg of phenylthiourea was added to each ml of TBS during the extraction procedure to inhibit cross linking reactions. The extracts were dialyzed against 2 changes of about 2 L of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl in preparation for hydroxyapatite batch chromatography. The samples were then filtered through glass ACRODISCS® (available from Gelman Sciences, Ann Arbor, Mich.) and added to 14 g of Macro-Prep Ceramic Hydroxyapatite, Type I, 40 μm beads (available from Bio-Rad), previously equilibrated in 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl. The extracts and beads were rocked at room temperature for about 30 minutes. Following incubation, the beads were centrifuged for about 5 minutes at 500×g and the supernatants removed. The beads were washed with about 40 ml 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl, centrifuged as above, and washed and centrifuged again to eliminate all unbound protein. Bound proteins were eluted by washing the beads with about 40 ml of each of 100 mM, 200 mM, 300 mM, and 400 mM phosphate buffer, pH 6.5 containing 10 mM NaCl. Following elution, the supernatants from each concentration of phosphate buffer were tested for juvenile hormone esterase activity as described previously in Example 7. The juvenile hormone esterase activity eluted at different phosphate concentrations in each batch, but the activity was generally found in the 200 mM to 300 mM phosphate fractions.
The fractions that contained the highest juvenile hormone esterase activity were combined and diafiltered into a total volume of about 50 ml of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl using a stirred cell concentrator fitted with a YM10 ultrafiltration membrane (available from Amicon, Beverly, Mass.). Aliquots of about 5 ml to 10 ml were applied to a chromatography column containing about 10 ml of Macro-Prep Ceramic Hydroxyapatite, Type I, 20 μm beads, previously equilibrated with 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl. The column was washed with 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl to 0.5 M 10 mM phosphate buffer, pH 6.5 containing 10 mM NaCl. Fractions were assayed for carboxylesterase activity using the assay described previously. The results indicated that carboxylesterase activity was eluted at about 160 mM phosphate.
The fractions that contained the highest carboxylesterase activity were combined and diafiltered into a total volume of about 15 ml of 20 mM sodium acetate buffer, pH 4.0 in preparation for cation exchange chromatography. Aliquots of about 3 ml were applied to a PolyCat A cation exchange column (available from PolyLC, Columbia, Md.) equilibrated in 20 mM sodium acetate buffer, pH 6.0, operated on a Waters high performance liquid chromatography system (available from Waters Corporation, Milford, Mass.). The column was washed with 20 mM sodium acetate buffer, pH 6.0 until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 20 mM sodium acetate buffer, pH 6.0 to 20 mM sodium acetate buffer, pH 6.0 containing 1 M NaCl. Fractions were assayed for CE activity using the assay described previously. The results indicated that there were two pools of CE activity. The first pool was not retained on the cation exchange column, and the second pool was eluted at about 170 mM NaCl.
The fractions from the second pool that contained the highest carboxylesterase activity were combined and diafiltered into a total volume of about 10 ml of 25 mM Tris (pH 8), 1 mM NaCl, in preparation for anion exchange chromatography. The sample was then applied to a Bio-Scale Q2 anion exchange column (available from Bio-Rad). The column was washed with 25 mM Tris (pH 8), 10 mM NaCl until all unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCl in 25 mM Tris, pH 8. Fractions were assayed for carboxylesterase activity using the assay described previously. The results indicated that carboxylesterase activity was eluted at about 350 mM NaCl.
Fractions containing carboxylesterase activity were combined and concentrated to about 175 ml using a Centricon 10 centrifugal concentrator (available from Amicon, Beverly, Mass.) in preparation for size exclusion chromatography. The sample was applied to a Bio-Select SEC 125-5 size exclusion chromatography column (available from Bio-Rad), previously equilibrated in TBS, pH 7.2. About 250 μl fractions were then collected. The fractions were assayed for carboxylesterase activity using the assay described previously. The results indicated that the carboxylesterase activity was eluted in about 5.5 to 6 ml of buffer, corresponding to a molecular weight of about 40 to 100 kDa based on the elution volumes of gel filtration molecular weight standard proteins (available from Sigma-Aldrich, St. Louis, Mo.).
Example 16This example describes the purification of juvenile hormone esterase activity from unfed adult flea midguts by affinity chromatography.
About 16,000 unfed adult flea midguts were collected in 20 mM Tris buffer (pH 7.7), containing 130 mM NaCl, 1 mM sodium EDTA, 1 mM PEFABLOC® (available from Boehringer Mannheim, Indianapolis, Ind.), 1 microgram/ml (μg/ml) leupeptin and 1 μg/ml pepstatin. The midguts were homogenized by freeze-fracture and sonication, and then centrifuged at about 14,000×g for 20 min. The soluble material from the centrifugation step was recovered, diafiltered into Tris buffered saline (TBS), and applied to a disposable plastic column containing about 1 ml of 3-[(4′-mercapto)butylthio]-1,1,1-trifluoropropane-2-one linked Sepharose 6B beads, prepared similarly to the method described by Venkatesh et al. (J. Biol. Chem., Vol. 265, No. 35, 21727-21732, 1990) (the 3-[(4′-mercapto)butylthio]-1,1,1-trifluoropropane-2-one was a gift from Novartis Corp., Basel, Switzerland; and the Epoxy-activated Sepharose 6B is available from Pharmacia Biotech Inc., Piscataway, N.J.). After overnight incubation at 4° C., the column was drained and the beads were washed with about 10 ml TBS, then about 10 ml TBS containing 0.1% (w/v) n-octylglucoside (OG; available from Boehringer Mannheim). The pre-column, flow-through, and wash fractions were tested for juvenile hormone esterase activity by the method previously described above in Example 7. The results indicate that the flow-through fraction contained approximately 40% less juvenile hormone esterase activity than the pre-column material, and that the washes contained very little activity.
Bound protein was eluted from the beads by adding about 10 ml of TBS containing 0.1% (w/v) OG and 1 mM 3-octylthio-1,1,1-trifluoropropane-2-one (OTFP; a gift from Novartis Corp.). After a 2 hour incubation at 4° C., about 5 ml of the eluate was collected, and the remaining 5 ml was incubated with the beads overnight at 4° C. The following day, the beads were drained, the eluate collected, and an additional 10 ml of TBS containing 0.1% (w/v) OG and 1 mM OTFP was added to the beads. After an overnight incubation at 4° C., the beads were drained and the eluate collected. The final 10 ml elution step was repeated 3 additional times so that we had 6 eluted fractions. The first elution fraction was dialyzed overnight twice against 1 liter of fresh TBS to remove excess OTFP. The second elution fraction was also dialyzed overnight against 1 liter of fresh TBS to remove OTFP. The third through sixth elution fractions were not dialyzed. All six eluted fractions were tested for juvenile hormone esterase activity by the method previously described above in Example 7. The results indicate that only the third elution fraction contained detectable juvenile hormone esterase activity. Analysis of the eluted fractions by silver-stained SDS-PAGE indicated that several proteins were specifically bound to the affinity beads and were eluted by OTFP. The apparent molecular weights of these proteins, as determined by SDS-PAGE, were about 66 kDa, 55 kDa, and 33 kDa. About 3.5 ml of each elution fraction were combined and concentrated to about 110 μl using a Centriplus 10 centrifugal concentrator (available from Amicon, Beverly, Mass.). This pool was separated by SDS-PAGE and blotted onto a polyvinylidene difluoride (PVDF) membrane as described previously in Example 5. The stained protein band at about 66 kDa was excised and subjected to N-terminal sequence analysis as described previously.
The results indicated that the N-terminal amino acid sequence of the putative 66 kDa juvenile hormone esterase protein was DL y/g V k/y/g v/q/n LQGTLKGKE (denoted herein as SEQ ID NO:74), in which the lower case letters designate uncertainties. Below is shown a comparison between different esterase amino acid sequences of the present invention.
This example describes the purification of an active recombinant juvenile hormone esterase protein from baculovirus supernatants.
About 1 liter of supernatant from cultures of S. frugiperda:pVL-nfE91600 cells producing the flea esterase protein PfE9528 was brought to about 50% saturation with ammonium sulfate and centrifuged at about 20000×g for about 30 minutes at 4° C. to pellet the precipitated material. After centrifugation, the pellet was retained and the supernatant was brought to about 100% saturation with ammonium sulfate and centrifuged as above. The material in both pellets were resuspended separately in about 35 ml of Tris buffered saline (TBS), pH 8.0. The resuspended pellets were assayed for the presence of flea esterase protein PfE9528 using standard Western blot techniques and a polyclonal antiserum that binds specifically to PfE9528 protein. Briefly, a rabbit was immunized with PHIS-PfE9528 protein purified from E. coli:pTrc-nfE91584 cells (described above in Example 12C) and boosted using standard procedures. The results indicated that the flea esterase protein PfE9528 was present in the S. frugiperda:pVL-nfE91600 supernatants and the protein was precipitated by adjusting the ammonium sulfate concentration from about 50% saturation to about 100% saturation.
The resuspended flea protein PfE9528 was diafiltered into about 10 ml of 25 mM Tris (pH 8.0), 10 mM NaCl using an Ultrafree-20 10 kD centrifugal concentrator in preparation for anion exchange chromatography. Aliquots of about 5 ml were loaded onto an Uno Q6 anion exchange column equilibrated in 25 mM Tris (pH 8.0), 10 mM NaCl. The column was washed with 25 mM Tris pH 8.0), 10 mM NaCl until most of the unbound protein was removed. Protein bound to the column was then eluted with a linear gradient from 10 mM to 1 M NaCl in 25 mM Tris buffer pH 8.0). Fractions were assayed for the presence of flea esterase protein PfE9528 by the immunoblot method described above. The results indicated that the flea esterase protein PfE9528 was eluted at about 200 mM NaCl.
Fractions containing the flea esterase protein PfE9528 were pooled and concentrated to about 440 μl using a Centricon 10 kD centrifugal concentrator in preparation for size exclusion chromatography. The sample was applied in 3 aliquots to a Bio-Select SEC 125-5 size exclusion chromatography column (available from Bio-Rad), previously equilibrated in TBS, pH 7.2. The column was eluted with TBS, pH 7.2 at a flow rate of about 0.5 ml/min, and fractions of about 250 μl were collected. The fractions were assayed for the presence of flea esterase protein PfE9528 by the immunoblot method described above. The results indicated that the flea esterase protein PfE9528 was eluted with about 6 ml of buffer, corresponding to a molecular weight of about 40 to 100 kDa based on the elution volumes of gel filtration molecular weight standard proteins (available from Sigma-Aldrich, St. Louis, Mo.).
Fractions containing flea esterase protein PfE9528 were then assayed for juvenile hormone esterase activity as described in Example 7 and carboxylesterase activity as described in Example 2. The results indicated that the purified flea esterase protein PfE9528 had both juvenile hormone esterase activity and carboxylesterase activity.
Claims
1-59. (canceled)
60. An isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule that encodes a protein at least 95% identical to a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:73, SEQ ID NO:74; and (b) an isolated nucleic acid molecule fully complementary to a nucleic acid molecule of (a).
61. The isolated nucleic acid molecule of claim 60, wherein said encoded protein has esterase activity.
62. A recombinant molecule comprising a nucleic acid molecule as set forth in claim 60 operatively linked to a transcription control sequence.
63. A recombinant virus comprising a nucleic acid molecule as set forth in claim 60.
64. A recombinant cell comprising a nucleic acid molecule as set forth in claim 60.
65. A method to produce a carboxylesterase protein, said method comprising culturing a cell capable of expressing said protein, said protein being encoded by a nucleic acid molecule of claim 60, part (a).
66. An isolated protein comprising an amino acid sequence at least 95% identical to a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:73, SEQ ID NO:74.
67. The isolated protein of claim 66, wherein said isolated protein has carboxylesterase activity.
68. The protein of claim 66, wherein said protein, when administered to an animal, elicits an immune response against a carboxylesterase protein.
69. A method to identify a compound capable of inhibiting flea carboxylesterase activity, said method comprising:
- (a) contacting a protein comprising an amino acid sequence at least 95% identical to a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:73, SEQ ID NO:74. with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has carboxylesterase activity; and
- (b) determining if said putative inhibitory compound inhibits said activity.
Type: Application
Filed: Jan 11, 2008
Publication Date: Jul 2, 2009
Inventors: Gary M. Silver (Fort Collins, CO), Nancy Wisnewski (Fort Collins, CO), Kevin S. Brandt (Fort Collins, CO)
Application Number: 12/013,262
International Classification: C12Q 1/44 (20060101); C12N 15/12 (20060101); C12N 5/10 (20060101); C12N 9/18 (20060101);
