GB2123005A - Bovine calf chymosin - Google Patents

Abstract

Cloned bovine calf chymosin and calf prochymosin genes, plasmids containing such cloned genes and microorganisms transformed by those plasmids are disclosed.

Description

SPECIFICATION Bovine calf chymosin Background of the invention The present invention relates to a cloned bovine gene that specifies the biosynthesis of bovine calf chymosin. The invention further relates to a plasmid containing a cloned bovine calf chymosin gene, as well as a microorganism transformed with such a plasmid.

Chymosin (also known as rennin (EC 3.2.23.4) is a proteolytic enzyme, that is abundant in the digestive tract of unweaned preruminant species. Chymosin is secreted as a zymogen, prochymosin, which is a single polypeptide chain containing 365 amino acids (37,000 daltons). Prochymosin is converted to chymosin by the specific removal of 42 N-terminal amino acids through a pH-dependent, autocatalytic reaction.

Bovine calf chymosin exists as two isozymes (designated A and B), which can be resolved by DEAE-cellulose chromatography of the crystalline enzyme. Isozyme B, which may be catalytically less efficient than isozyme A, is also the more abundant form in tissue extracts. Bovine calf chymosin isozyme B has been sequenced, and isozyme A has been partially sequenced, to reveal only one amino acid difference at residue 290 (glycine in B and aspartic acid in A) (see Foltmann, B. et al., J. Biol.

Chem., 254: 8447-8456 [1976]).

Bovine calf chymosin (mixture of isozymes) has been used for centuries in the manufacture of cheese. However, since World War II, a shortage of calf chymosin has developed, largely as a result of a decline in veal consumption. Accordingly, the chymosin obtained from abomasal tissue (rennet) of unweaned calves has steadily been replaced by other enzymes of animal and microbial origin; none of which possess the optimal characteristics of the calf enzyme.

As a result of this shortage, there has been considerable interest in bacterially cloning a bovine calf chymosin gene and developing a chymosin-producing microorganism. Various investigators have reported the extraction of chymosin mRNA from bovine abomasal tissue (see H.

Uchiyama, et ai.,Agric. Biol. Chem., 44, 1373-1381 [1980]), and the cloning of a double-stranded cDNA (ds-cDNA) chymosin gene in E. colt (see Beppu, T., et al., Recombinant DNA Industrial Applications (The Cetus Symposium) IFS Meeting, p. F-21.5(L) [1980]). Nishimori, K. et al. (J.

Biochem., 90,901-904 [1981]) describe attempts to clone a prochymosin gene inE. coil and report obtaining a clone containing a 1021 base pair insert that apparently has sequence homoiogy with the bovine chymosin gene (based on hybrid arrested translation observations). This 1021 base pair insert represents about 80% of the full length chymosin gene, assuming that the entire segment is derived from the chymosin gene. However, the authors report no characterizing data for the gene, such as sequence information or a restriction map, therefore, it is not known to what degree the cloned fragment is derived from the bovine calf chymosin gene. Moreover, the authors do no report observing any protein expression by their bacterial clones.

There is, therefore, a continuing need for a cloned, full-length bovine calf chymosin gene and a microbial strain capable of producing bovine calf chymosin.

Summary of the invention The present invention concerns a cloned, intact bovine calf chymosin gene, which may be comprised in a cloned bovine calf prochymosin gene, plasmids containing such genes and microorganisms transformed with such plasmids. In addition, a method is disclosed for producing bovine calf chymosin or prochymosin, which involves cultivating a calf prochymosin-producing microorganism on a nutrient medium under conditions yielding chymosin or prochymosin respectively.

Detailed description of the invention The procedures for obtaining and cloning a bovine calf chymosin gene described herein are, except where otherwise indicated, accomplished by using conventional techniques of molecuiar biology. E.g. see Ullrich, A. et al., Science, 196, 1313 (1977) and Seeburg, P. H. et al., Nature, 270, 486 (1977).

The procedures leading to a bovine calf chymosin-producing microorganism can be divided into the following major stages, each of which is described more fully herein: (1) extraction of prochymosin mRNA from the mucosal lining of the fourth stomachs of milk-fed calves, (2) in vitro synthesis of double-stranded cDNA, using bovine calf prochymosin mRNA as a template, (3) insertion of the double-stranded cDNA into a suitable cloning vector and transformation of cells with that cloning vector, and (4) selection of microbial clones containing the bovine calf prochymosin gene based on their ability to express bovine prochymosin. The experimental details of these techniques are discussed below in the "Examples" section; however, it is understood that, in light of the present disclosure, these techniques can be modified substantially, while still obtaining the desired results.

Bovine prochymosin mRNA is advantageously recovered as polysomal mRNA from comminuted mucosal lining of the fourth stomachs of milk-fed calves. Ribonucleases are present in this tissue in relatively high concentrations, therefore, the tissue is preserved by freezing shortly following the death of the animal, and substances are employed during the extraction steps that reduce ribonuclease activity. Generally, the procedure of C. Vaslet et al., Nature, 285, 674-676 (1980), may be employed for extracting polysomal mRNA. Frozen tissue can be pulverized and homogenized and, after a preliminary centrifugation to remove lipids and insoluble cell debris, the relatively dense polysomal mRNA is advantageously separated by centrifugation through a sucrose gradient.Polyadenylated mRNA can conveniently be recovered from the polysomal mRNA preparation, by elution over an oligo d(T)-cellulose column. Green, M. et al., Arch. Biochem. Biophys., 172, 74 (1976).

To obtain an enriched preparation of prochymosin mRNA, the eluate from the oligo d(T)-cellulose column can be fractionated by centrifugation in a sucrose gradient, as is well known in the art. The mRNA fractions obtained from the sucrose gradient centrifugation are advantageously tested for prochymosin mRNA content by determining their translational activity in a cell-free translation system.

A number of cell-free translational systems have been devised, such as wheat germ extract (Martial, J.

et al., Proc. Nat'IAcad. Sci. USA, 74, 1816 (1977)), an mRNA-dependent retriculocyte lysate (Pelham, H. R. B. et al., Eur. J. Biochem., 67, 247 (1976)), andoocytes from Xenopus laevis (Sloma, A. et al., Methods in Enzymology, Vol. 70 (1 981)). The wheat germ extract system is preferred for the testing of prochymosin mRNA. The wheat germ cell-free system can be supplemented with a radioactively labeled amino acid, such as S35-methionine, so that the resulting proteins contain a tracer. The various wheat germ products are then conveniently tested for prochymosin content by gel electrophoresis followed by visualization by fluorography, and the mRNA fraction found to produce prochymosin in this system is used for ds-cDNA synthesis.

Synthesis of cDNA employs avian myeloblastosis virus reverse transcriptase. This enzyme catalyzes the synthesis of a single strand of DNA from deoxynucleoside triphosphates on the mRNA template. Kacian, D. L., et al.,Proc. Nat'lAcad. Sci. USA, 73, 2191 (1976).The poly r(A) tail of mRNA permits oligo (dT) (of about 12-1 8 nucleotides) to be used as a primer for cDNA synthesis. The use of a radioactively-labelled deoxynucleoside triphosphate facilitates monitoring of the synthesis reaction.

Generally, a 32P-containing deoxynucleoside triphosphate, such as [a!-32P]dCTP may be used advantageously for this purpose. The cDNA synthesis is generally conducted by incubating a solution of the mRNA, the deoxynucleoside triphosphates, the oligo (dT) and the reverse transcriptase. The solution also preferably contains small amounts of actinomycin D and dithiothreitol to promote fuil length synthesis. Kacian, D. L., et al. supra. After incubation, ethylenediaminetetraacetic acid is added to the solution, and the solution is extracted with phenol:chloroform. The aqueous phase is advantageously purified by gel filtration chromatography, and the cDNA-mRNA complex in the eluate is precipitated with alcohol.

The mRNA can be selectively hydrolyzed in the presence of the cDNA with dilute sodium hydroxide at an elevated temperature. Neutralization of the alkaline solution and alcohol precipitation yields a single-stranded cDNA copy.

The single-stranded cDNa copy has been shown to have a 5'-poly (dT) tail, and to have a 3' terminal hairpin structure, which provides a short segment of duplex DNA. Efstratiadis, A., et al., Cell, 7, 279 (1976). This 3' hairpin structure can act as a primer for the synthesis of a second DNA strand.

Synthesis of this second strand is conducted under essentially the same conditions as the synthesis of the cDNA copy, except that the Klenow fragment of E. coli DNA polymerase I (Klenow, H., et al., Eur. J.

Biochem., 22, 371 (1971)) is substituted for reverse transcriptase. The duplex cDNA recovered by this procedure has a 3' loop, resulting from the 3' hairpin structure of the single-stranded cDNA copy. This 3' loop can be cleaved by digestion with the enzyme, S1 nuclease, using essentially the procedure of Ullrich, A., et al., supra. The S1 nuclease digest may be extracted with phenol-chloroform, and the resulting ds-cDNA precipitated from the aqueous phase with alcohol.

For purposes of amplification and selection, the ds-cDNA gene prepared as described above is generally inserted into a suitable cloning vector, which is used for transforming appropriate host cells.

Suitable cloning vectors include various plasmids and phages, but plasmids are generally preferred.

The criteria for selecting a cloning vector include its size, its capability for replicating in the host cells, the presence of selectable genes, and the presence of a site for insertion of the gene. With respect to its size, the vector is advantageously relatively small, to permit large gene insertions, and so as not to divert large amounts of cellular nutrients and energy to the production of unwanted macromolecules.

The vector also includes an intact replicon which remains functional after insertion of the gene. This replicon preferably directs the desired mode of replication of the plasmid, i.e., multiple copies or a single copy per cell, or a controllable number of copies per cell. Genes specifying one or more phenotypic properties, preferably antibiotic resistance, facilitate selection of transformants. The insertion site is advantageously a unique restriction site for a restriction endonuclease. A cloning vector meeting all of these criteria is the plasmid pBR322. Bolivar, F., et al. Gene, 2, 95 (1977). This plasmid is small (about 2.8 x 106 daltons), carries genes for ampicillin (amp) and tetracycline (tet) resistance, and is subject to relaxed replication in E. coli. The plasmid also has a unique restriction site for the endonuclease, Pstl, which occurs within the amp gene. The ds-cDNA can be conveniently inserted into this plasmid by a homopolymeric tailing technique. Nelson, T., et al., Methods of Enzymology, 68, 41 (1980). Homopolymer tails, e.g., polydC, are added to the 3'-hydroxyls of the ds-cDNA, by reaction with the appropriate deoxynucleoside triphosphate, e.g., dCTP, in the presence of terminal deoxynucleotidyl transferase (Chang, L. M. S., et al., J. Biol. Chem., 246, 909 (1971)). Poly d(C) tails ds-cDNA's are preferably sized on neutral sucrose gradients (see Norgard et al., J. Biol. Chem., 255, 7665-7672 (1980)). Double-stranded cDNA's longer than 600 base pairs are employed in subsequent steps.

The plasmid is opened by digestion with the appropriate endonuclease, and complementary homopolymeric tails, e.g., poly dG, are added to the 3'-hydroxyls of the opened plasmid, using the identical homopolymer tailing technique, e.g., using dGTP. If desired, the tailing reactions may be monitored, by employing radioactively labelled deoxynucleotide triphosphates e.g., [3H]dCTP and [3H]dGTP, in the reactions. Generally, the reactions are conducted to provide tails about 1 0-20 nucleotides long. The tailed cDNA and plasmid are recovered, e.g., by phenol extraction followed by alcohol precipitation. The two "tailed" DNA species are annealed by incubating a buffered solution of equimolar concentrations of the two species, to yield a recombinant plasmid containing the bovine calf prochymosin gene.

A suitable amps, tets strain of E. coli may be transformed with the recombinant plasmid, using essentially the method of Lederberg, J. Bacteriology, 119, 1072 (1974). Transformants are typically grown on a standard L-broth, containing tetracycline. Samples of colonies growing on the tetracyclinecontaining medium are then transferred to a second medium containing ampicillin. Because the pBR322 plasmid imparts tetracycline resistance to the cells, colonies growing on the tetracyclinecontaining medium must contain that plasmid. On the other hand, the ampicillin resistance of the pBR322 plasmid is destroyed by insertion of the gene, therefore, only tetR, amps colonies are selected for further analysis.

Generally, several hundred to several thousand potential prochymosin clones are produced by these procedures. To identify a smaller group of colonies which contain the prochymosin gene, a radioactively labelled DNA probe may advantageously be employed. Grunstein, M, et al., Proc. Nat'l, Acad. Sci. USA, 72, 3961-3965 (1975). Such a DNA probe can advantageously be prepared from a highly enriched prochymosin mRNA population, using reverse transcriptase and incorporating a radiolabeled nucleotide. Generally, the method of Grunstein et al. (supra) as modified by Wahl et al., Proc. Nat. Acad. Sci., USA 76,3683-3687 (1979), is a preferred method for conducting the colony hybridization analysis.

Although colony hybridization generally reduces the number of potential prochymosin clones a considerable number of such clones usually still exists. A procedure which has been employed successfully for further screening of such clones involves an enzyme-linked immunosorbent assay (ELISA). This assay detects chymosin in those clones where protein expression occurs. The assay involves coating chymosin standards and cell lysates onto the inner surfaces of wells of a plastic microtiter plate, and immunologically reacting any chymosin present with anti-chymosin antibody. This immobilized conjugate is then reacted with an enzyme-tagged second antibody that is specific for the first antibody. After separating unbound tagged antibody, a chromogenic substrate is introduced to determine the bound enzyme present, which is a measure of the level of chymosin in the original cell lysate.This technique has been found very useful for isolating a few clones from a large population of clones identified as positive by colony hybridization analysis.

The few clones identified by the enzyme-linked immunoassay system are advantageously further characterized by restriction mapping and sequencing. One clone, designated pGx 1225, has given consistently strong positive results by ELISA, and also has been confirmed to contain the prochymosin gene by sequence analysis and restriction mapping. The restriction map of the prochymosin gene insert of pGx 1225 is shown in Figure 1 of the drawings. The insert consists of 1289 base pairs. The zymogen is specified by the region from base pair 43 through base pair 1137 and the mature chymosin gene is specified by base pair 1 69 through base pair 1137.

The nucleotide sequence of this prochymosin gene insert was determined by the method of Sanger, et al. Proc. Nat'lAcad. Sci. USA, 74, 5463-5467 (1977), and this nucleotide sequence is shown in Figure 2 of the drawings. The drawing shows the 5' - - < 3' strand of the noncoding and coding regions, along with the amino acid sequence specified by the gene.As used in Figure 2 and elsewhere herein, the abbreviations have the following standard meanings: A=deoxyadenyl T=thymidyl G=deoxyguanyl C=deoxycytosyl G LY=glycine ALA=alanine VAL=valine LEU=leucine ILE=isoleucine SER=serine THR=threonine PHE=phenylalanine TYR=tyrosine TRP=tryptophan CYS=cysteine MET=methionine ASP=aspartic acid GLU=glutamic acid LYS=lysine ARG=arginine HlS=histidine PRO=proline GLN=glutamine ASN=asparagine It will be appreciated that because of the degeneracy of the genetic code, the nucleotide sequence of the gene can vary substantially.For example, portions oral of the gene could be chemically synthesized to yield DNA having a different nucieotide sequence than that shown in Figure 2, yet the amino acid sequence would be preserved, provided that the proper codon-amino acid assignments were observed. Having established the nucleotide sequence of the bovine calf prochymosin gene and the amino acid sequence of the protein, the gene of present invention is not limited to a particular nucleotide sequence, but includes all variations thereof as permitted by the genetic code.

A culture of E. coli cells, pGx 1225, containing the prochymosin gene insert described above has been deposited with the U.S. Department of Agriculture, Northern Regional Research Laboratory, Peoria, Illinois, U.S.A., as NRRL No. 8-15061.

The present invention has been described in connection with the use of E. coli as the bacterial host for recombinant DNA containing the bovine calf prochymosin gene, but skilled molecular biologists will appreciate that other gram-negative bacteria, such as Pseudomonas; gram-positive bacteria, such as Bacillus; and higher unicellular organisms, such as yeasts and fungi can be employed for cloning and/or expression of the prochymosin gene or portions thereof.

The invention is further illustrated by reference to the following examples, which are not intended to be limiting.

Example I Preparation of polysomal mRNA The fourth stomachs of milk fed calves were collected at a local abattoir (Henry Stapf, Inc., Baltimore, Maryland) within thirty minutes after the animal was sacrificed. The mucosal lining was removed and quick-frozen in liquid nitrogen for storage. Polysomal mRNA was prepared according to a modification of the procedure described by Vaslet et al. (Nature 285: 674--676 [1980]). Pulverized frozen tissue (50 grams) was homogenized in 100 milliliters of buffer containing 0.25 M KCI, 0.025 M MgCl2, 0.05 M tris-HCI pH 7.4, 0.5% NP40, 200 4g/ml cyclohexamide, and 250 yg/mi heparin. The homogenate was twice centrifuged at 10,000 rpm for 10 minutes at 40C to clear a heavy lipid layer and remove cell debris.Polysomes were collected as a pellet prepared by centrifuging the low speed supernatant through a 2.5 ml cushion of 52% sucrose in homogenization buffer lacking NP40, cyclohexamide and heparin at 28,000 rpm for 8 hours at 40C in a Beckman SW28 rotor. The polysomal pellet was resuspended in 50 mM tris-HCI pH 9, 1 mM EDTA, 1 50 mM NaOAc, 1% SDS containing proteinase K (250 yg/ml). Following a 60 minute incubation at 370C, the preparation was extracted two times with phenol:chloroform and the RNA was alcohol precipitated at O to -800C for 1 hour. The RNA was pelleted and washed 3 times with 3 M sodium acetate to solubilize small RNAs, mucin, and heparin. The final polysomal RNA pellet was resuspended in deionized water and stored at -800C.

Poly(A)+mRNA was obtained by hybridization of polysomal RNA to an oligo d(T)-cellulose column. The column was equilibrated with binding buffer containing 10 mM tris-HCI pH 7.6, 1 mM EDTA, 0.1% SDS, and 400 mM NaCI, and the whole polysomal RNA preparation was cycled through the column 5 times. Unbound RNA was eluted with several column volumes of binding buffer. Elution buffer containing 10 mM tris-HCI pH 7.6, 1 mM EDTA, 0.1% SDS was used to wash bound poly(A)+mRNA from the column. The poly(A)+ mRNA was alcohol precipitated and stored at -800C.

Example II Sucrose gradient fractionation of Poly(A)+mRNA Poly(A)+ mRNA was layered on a 5%20% continuous sucrose density gradient made up in SDS buffer containing 0.5% SDS, 0.1 M NaCI, 0.01 Mtris pH 7.5, 0.001 M EDTA. The RNA was heated to 8.00C for 2 minutes and quick cooled on ice just before layering on the gradient. Calf rennet rRNAs were run in separate tubes as sedimentation standards. The gradients were spun at 30,000 rpm for 20 hours at 220C in a Beckman SW40 rotor, and 0.6 ml fractions were collected to generate an A260 profile, which is shown in Figure 3 of the drawings.The mRNA in each fraction was alcohol precipitated and subsequently tested for translational activity in a commercially prepared wheat germ system (BRL) (see Example Ill). The shaded fraction shown in Figure 3 was enriched for bovine prochymosin mRNA and was used for ds-cDNA synthesis (Example IV).

Example Ill Wheat germ translation of mRNAs and polyacrylamide gel electrophoresis RNA samples were translated in a wheat germ cell-free system supplemented with S35- methionine as suggested by the supplier. (Bethesda Research Laboratory, Rockville, Maryland). The wheat germ products were electrophoresed in polyacrylamide-SDS slab gels containing a 5% stacking and 12.5% separating gel (Laemmli, Nature 227: 680-685 [1970]). The radiolabeled proteins were visualized by fluorography (Bonner and Laskey, Eur. J. Biochem. 46: 83-88 [1 974]) with Kodak XAR5 film.

Example IV Preparation of hybrid plasmids Double-stranded cDNA synthesis and homopolymeric tailing reactions were performed with only slight modification as detailed in McCandliss etna!. (Methods of Enzymology, Vol. 70, 1 981).

Approximately 1 0-20 dG residues were added to the 3' end of Pstl linearized pBR322. The same number of dC residues were added to the synthesized ds-cDNA. C-tailed ds-cDNA was sized on neutral sucrose gradients as outlined by Norgard etna!. (JBC 255: 7665-7672 [1980]). The ds-cDNA was dissolved in distilled H2O and layered on a chilled 12 ml 5%25% sucrose gradient containing 50 mM tris-HCI pH 7.5 and 1 mM EDTA. The gradient was spun at 38,000 rpm for 17.5 hours at 5"C in a Beckman SW40 rotor. A second gradient containing 10 y9 of Taql endonuclease digested plasmid pBR322 was run simultaneously for use as size standards.The gradients were fractionated and analyzed as follows: a) Taql digested pBR3 22 gradient. The DNA in each 0.5 ml fraction was alcohol precipitated and resuspended in sample buffer containing 0.9 M tris-HCI, pH 8.5, 0.09 M Boric acid, 40 mM EDTA, 5% glycerol, 0.025% xylene cyanol, 0.025% bromphenyl blue. Each sample was electrophoresed on an 8% acrylamide slab gel. The ethidium bromide stained gel was photographed and lanes cohtaining DNA greater than 600 base pairs were noted. b) ds-cDNA gradient - 1 00 Ml of each 1 ml fraction was added to scintilate for counting.The DNA in each fraction was alcohol precipitated and those containing ds-cDNA longer than 600 base pairs (as indicated by the Taql digested pBR322 gel information) were employed in subsequent steps.

The oligo d(G)-tailed pBR322 DNA and sized oligo d(C)-tailed ds-cDNA were diluted to equimolar concentrations in 10 mM tris-HCI pH 7.5, 100 mM NaCI, 2.5 mM Na2 EDTA. The DNAs were annealed at 700C for 10 minutes and then cooled slowly overnight at room temperature.

Example V Transformation Recombinant plasmids were introduced into E. coli HB101 substantially according to the method of Lederberg and Cohen (J. Bacti. 119: 1072 [1974]). Transformants were grown on standard LBtetracycline (15 yg/ml) plates at 37 OC for 24 hours. Colonies were picked to LB-ampicillin (50 yg/ml) plates to determine phenotype. Only ampicillin sensitive colonies were selected for further analysis.

Approximately 750 clones were isolated.

Example VI Colony hybridization Transformants were prepared for colony hybridization according to a modification of the procedure originally described by Grunstein and Hogness (PNAS, 72: 3961-3965 [1975]). The DNA bound filters were hybridized in the presence of dextran sulfate and formamide as suggested by Wahl eft at (PNAS 76: 3683-3687 [1979]). The filters were pre-hybridized in 6 xSSC (20X=3, M NaCI; 0.3 MNa3 Citrate); 1 xDenhardts' solution (10X-0.2% BSA; 0.2% PVP-4O; 0.2% Ficoll); 0.1% SDS in a glass jar with gentle swirling at 420C for 60 minutes.A second pre-hybridization followed in a 8 mls/filter of 50%formamide,5xSSC,5xDenhardts' solution, 50 mM NaPO4pH 6.5, 1% glycine,195 ug/ml denatured salmon sperm DNA for 2 hours 420C with gentle swirling. A 32p-cDNA probe was added (=0.5x106 cpm/filter) to a hybridization mix containing 50%formamide, 5xSSC, 1 xDenhardt's solution, 20 mM NaPO4 pH 6.5, 10% dextran sulfate, 100 MG/ml denatured salmon sperm DNA and incubated with the filters at 42 OC for 18 hours with gentle swirling. This probe was prepared from a sucrose gradient fraction highly enriched for prochymosin mRNA (Example II) that was the next higher adjacent fraction to that used for ds-cDNA synthesis.The filters were washed 3 xl 0 minutes in 2 x SSC, 0.1% SDS at room temperature and 2x10 minutes in 0.1 xSSC, 0.1% SDS at 500C. Positives were visualized by exposing the blotted filters to Kodak XR-5 film overnight at -8O0C with an intensifying screen.

Example VII Enzyme-linked immunosorbent assay The enzyme-linked immunoassorbent assay (ELISA) introduced by Engvall, E. and Pearlman, P.

(lmmunochemistry, 8: 871-874 [1971]) was used to screen the 135 recombinant clones which had previously been identified as positive by the procedure of Example VI. Antibody preparation: Chymosin (Sigma) was re-purified on an isoelectric focusing column and the purified chymosin was used as the immunogen in all subsequent injections. New Zealand white rabbits were initially immunized with 1 mg of chymosin in Complete Freund's Adjuvant administered intramuscularly. Boosters contained 0.5-0.75 mg chymosin in Incomplete Freund's Adjuvant and were similarly administered at bimonthly intervals for three months. Blood samples were then taken and used to prepare antichymosin serum.

Assay: Overnight cultures of colonies to be screened were set up in 5 mls of L-broth containing 25 mg/liter of tetracycline. Cultures of Hub101 containing PBR322 were used as a negative control. The cultures were spun down in the Sorval SM-24 rotor for 5 minutes, 40C, 8,000 RPM. Cells were washed once in phosphate-buffered saline (PBS), transferred to eppendorf tubes and resuspended in 300 ml of 20 mM carbonate/bicarbonate buffer pH 9.6 and placed on ice. Each tube was sonicated on ice for 10-1 5 seconds at 200 watts. The debris was pelleted and the lysate was carefully transferred to a clean eppendorf tube. 200 ul of cell lysate was added to each well of a microtiter plate (Linbro, Flow Labs).Three sets of standard curves ranging from 1 ,ug/ml-0.1 ,ug/ml in two-fold dilutions were made with re-purified chymosin. Each standard (200 yI) was added to the appropriate wells of the microtiter plate which was incubated overnight at 40C. The cell lysates and standards were aspirated from the plate and washed three times with PBS-.05% Tween 20.

Two hundred microliters of 3% rabbit serum albumin was added to each well and incubated at 370C for one hour to block remaining active binding sites on the plastic. The plate was washed three times with PBS-Tween and 200 ul of anti-chymosin antibody (diluted 1/32) was added to each well with the exception of one set of standards which received pre-immune serum diluted 1/32. The plate was incubated at 370C for 1 hour and washed three times with PBS-Tween. 200 ul of goat-anti-rabbit antibody conjugated with horseradish peroxidase (Sigma) and diluted 1/500 was added to each well and incubated for 1 hour at 370C. After washing three times with PBS-Tween 200 ul of the substrate, 2,2'-azino-di[3-ethylbenzthiozolin-sulfonate] (ABTS), was added to each well. The plate was read on a Flow Multi-Scanner at 405 nm 1 5 minutes after the addition of the substrate. Two clones were identified by this procedure as giving consistently positive results. A plasmid from one of these clones designated pGx1 225, contained an insert of sufficient length to code for a complete bovine calf prochymosin gene. This was isolated by gel electrophoresis as described by Sanger et al (Proc. Nat Acad. Sci., USA.. 74 5463-5467 (1977)). The DNA of this insert was characterised by restriction mapping and sequence analysis, and a culture of the cells was deposited with the U.S. Department of Agriculture, Northern Regional Research Laboratory, Peoria, Illinois as NRRL No. B-1 5061.

Claims (20)

Claims

1. A cloned bovine calf chymosin gene.

2. A cloned bovine calf chymosin gene as claimed in claim 1, comprising a cloned bovine calf prochymosin gene.

3. The bovine calf chymosin gene of claim 1, comprising the following deoxyribonucleotide sequence; Gly Glu Val Ala Ser Val Pro Leu GGX GAM GTX GCX QRS GTX CCX YTZ 169 Thr Asn Tyr Leu Asp Ser Gln Tyr ACX AAY TAY YTZ GAY ORS CAM TAY 193 Phe Gly Lys lle Tyr Leu Gly Thr TTY GGX AAM ATH TAY YTZ GGX ACX 217 Pro Pro Gln Glu Phe Thr Val Leu CCX CCX CAM GAM TTY ACX GTX YTZ 241 Phe Asp Thr Gly Ser Ser Asp Phe TTY GAY ACX GGX ORS QRS GAY TTY 265 Trp Val Pro Ser lle Tyr Cys Lys TGG GTX CCX ORS ATH TAY TGY AAM 289 Ser Asn Ala Cys Lys Asn His Gln QRS AAY GCX TGY AAM AAY CAY CAM 313 Arg Phe Asp Pro Arg Lys Ser Ser LGN TTY GAY CCX LGN AAM QRS QRS 337 Thr Phe Gln Asn Leu Gly Lys Pro ACX TTY CAM AAY YTZ GGX AAM CCX 361 Leu Ser lle His Tyr Gly Thr Gly YTZ ORS ATH CAY TAY GGX ACX GGX 385 Ser Met Gln Gly lle Leu Gly Tyr QRS ATG CAM GGX ATH YTZ GGX TAY 409 Asp Thr Val Thr Val Ser Asn lle GAY ACX GTX ACX GTX QRS AAY ATH 433 Val Asp lle Gln Gln Thr Val Gly GTX GAY ATH CAM CAM ACX GTX GGX 457 Leu Ser Thr Gln Glu Pro Gly Asp YTZ QRS ACX CAM GAM CCX GGX GAY 481 Val Phe Thr Tyr Ala Glu Phe Asp GTX TTY ACX TAY GCX GAM TTY GAY 505 Gly lle Leu Gly Met Ala Tyr Pro GGX ATH YTZ GGX ATG GCX TAY CCX 529 Ser Leu Ala Ser Glu Tyr Ser lie QRS YTZ GCX QRS GAM TAY QRS ATH 553 Pro Val Phe Asp Asn Met Met Asn CCX GTX TTY GAY AAY ATG ATG AAY 577 Arg His Leu Val Ala Gln Asp Leu LGN CAY YTZ GTX GCX CAM GAY YTZ 601 Phe Ser Val Tyr Met Asp Arg Asn TTY ORS GTX TAY ATG GAY LGN AAY 625 Gly Gln Glu Ser Met Leu Thr Leu GGX CAM GAM QRS ATG YTZ ACX YTZ 649 Gly Ala lle Asp Pro Ser Tyr Tyr GGX GCX ATH GAY CCX QRS TAY TAY 673 Thr Gly Ser Leu His Trp Val Pro ACX GGX QRS YTZ CAY TGG GTX CCX 697 Val Thr Val Gln Gln Tyr Trp Gln GTX ACX GTX CAM CAM TAY TGG CAM 721 Phe Thr Val Asp Ser Val Thr lle TTY ACX GTX GAY QRS GTX ACX ATH 745 Ser Gly Val Val Val Ala Cys Glu QRS GGX GTX GTX GTX GCX TGY GAM 769 Gly Gly Cys Gln Ala lle Leu Asp GGX GGX TGY CAM GCX ATH YTZ GAY 793 Thr Gly Thr Ser Lys Leu Val Gly ACX GGX ACX QRS AAM YTZ GTX GGX 817 Pro Ser Ser Asp lle Leu Asn lle CCX QRS QRS GAY ATH YTZ AAY ATH 841 Gln Gln Ala lle Gly Ala Thr Gln CAM CAM GCX ATH GGX GCX ACX CAM 865 Asn Gln Tyr Gly Glu Phe Asp lle AAY CAM TAY GGX GAM TTY GAY ATH 889 Asp Cys Asp Asn Leu Ser Tyr Met GAY TGY GAY AAY YTZ QRS TAY ATG 913 Pro Thr Val Val Phe Glu lle Asn CCX ACX GTX GTX TTY GAM ATH AAY 937 Gly Lys Met Tyr Pro Leu Thr Pro GGX AAM ATG TAY CCX YTZ ACX CCX 961 Ser Ala Tyr Thr Ser Gln Asp Gln QRS GCX TAY ACX QRS CAM GAY CAM 985 Gly Phe Cys Thr Ser Gly Phe Gln GGX TTY TGY ACX QRS GGX TTY CAM 1009 Ser Glu Asn His Ser Gln Lys Trp QRS GAM AAY CAY QRS CAM AAM TGG 1033 lle Leu Gly Asp Val Phe lle Arg ATH YTZ GGX GAY GTX TTY ATH LGN 1057 Glu Tyr Tyr Ser Val Phe Asp Arg GAM TAY TAY QRS GTX TTY GAY LGN 1081 Ala Asn Asn Leu Val Gly Leu Ala GCX AAY AAY YTZ GTX GGX YTZ GCX 1105 Lys Ala lle AAM GCX ATH 1129 wherein, the t' to 3' strand, beginning with the amine terminus and the amino acids for which each triplet codes are shown, and wherein within each triplet, A is deoxyadenyl T is thymidyl G is deoxyguanyl C is deoxycytosyl XisA,T,CorG Y is T or C When Y is C, Z is A, T, C or G WhenYisT,ZisAorG H isA,TorC QisTorA WhenQisT,RisCand SisA,T,CorG When Q is A, R is G and S is T or C M isAorG LisAorC When LisA, N isAorG When L is C, N is A, T, C orG G-LY is glycine ALA is alanine VAL is valine LEU is leucine ILE is isoleucine SER is serine THR is threonine PHE is phenylalanine TYR is tyrosine TRP is tryptophan CYS is cysteine MET is methionine ASP is aspartic acid GLU is glutamic acid LYS is lysine ARG is arginine HIS is histidine PRO is proline GLN is glutamine ASN is asparagine

4.The bovine calf prochymosin gene of claim 2, comprising the following deoxyribonucleotide sequence: Ala Glu GCX GAM 43 lle Thr Arg lle Pro Leu Tyr Lys ATH ACX LGN ATH CCX YTZ TAY AAM 49 Gly Lys Ser Leu Arg Lys Ala Leu GGX AAM QRS YTZ LGN AAM GCX YTZ 73 Lys Glu His Gly Leu Leu Glu Asp AAM GAM CAY GGX YTZ YTZ GAM GAY 97 Phe Leu Gln Lys Gln Gln Tyr Gly TTY YTZ CAM AAM CAM CAM TAY GGX 121 lle Ser Ser Lys Tyr Ser Gly Phe ATH QRS QRS AAM TAY QRS GGX TTY 145 Gly Glu Val Ala Ser Val Pro Leu GGX GAM GTX GCX QRS GTX CCX YTZ 169 Thr Asn Tyr Leu Asp Ser Gln Tyr ACX AAY TAY YTZ GAY QRS CAM TAY 193 Phe Gly Lys lle Tyr Leu Gly Thr TTY GGX AAM ATH TAY YTZ GGX ACX 217 Pro Pro Gln Glu Phe Thr Val Leu CCX CCX CAM GAM TTY ACX GTX YTZ 241 Phe Asp Thr Gly Ser Ser Asp Phe TTY GAY ACX GGX QRS QRS GAY TTY 265 Trp Val Pro Ser lle Tyr Cys Lys TGG GTX CCX ORS ATH TAY TGY AAM 289 Ser Asn Ala Cys Lys Asn His Gln QRS AAY GCX TGY AAM AAY CAY CAM 313 Arg Phe Asp Pro Arg Lys Ser Ser LGN TTY GAY CCX LGN AAM QRS QRS 337 Thr Phe Gln Asn Leu Gly Lys Pro ACX TTY CAM AAY YTZ GGX AAM CCX 361 Leu Ser lle His Tyr Gly Thr Gly YTZ QRS ATH CAY TAY GGX ACX GGX 385 Ser Met Gln Gly lle Leu Gly Tyr QRS ATG CAM GGX ATH YTZ GGX TAY 409 Asp Thr Val Thr Val Ser Asn lle GAY ACX GTX ACX GTX QRS AAY ATH 433 Val Asp lle Gln Gln Thr Val Gly GTX GAY ATH CAM CAM ACX GTX GGX 457 Leu Ser Thr Gln Glu Pro Gly Asp YTZ QRS ACX CAM GAM CCX GGX GAY 481 Val Phe Thr Tyr Ala Glu Phe Asp GTX TTY ACX TAY GCX GAM TTY GAY 505 Gly lle Leu Gly Met Ala Tyr Pro GGX ATH YTZ GGX ATG GCX TAY CCX 529 Ser Leu Ala Ser Glu Tyr Ser lle QRS YTZ GCX QRS GAM TAY QRS ATH 553 Pro Val Phe Asp Asn Met Met Asn CCX GTX TTY GAY AAY ATG ATG AAY 577 Arg His Leu Val Ala Gln Asp Leu LGN CAY YTZ GTX GCX CAM GAY YTZ 601 Phe Ser Val Tyr Met Asp Arg Asn TTY QRS GTX TAY ATG GAY LGN AAY 625 Gly Gln Glu Ser Met Leu Thr Leu GGX CAM GAM QRS ATG YTZ ACX YTZ 649 Gly Ala lle Asp Pro Ser Tyr Tyr GGX GCX ATH GAY CCX QRS TAY TAY 673 Thr Gly Ser Leu His Trp Val Pro ACX GGX ORS YTZ CAY TGG GTX CCX 697 Val Thr Val Gln Gln Tyr Trp Gln GTX ACX GTX CAM CAM TAY TGG CAM 721 Phe Thr Val Asp Ser Val Thr lle TTY ACX GTX GAY QRS GTX ACX ATH 745 Ser Gly Val Val Val Ala Cys Glu QRS GGX GTX GTX GTX GCX TGY GAM 769 Gly Gly Cys Gln Ala lle Leu Asp GGX GGX TGY CAM GCX ATH YTZ GAY 793 Thr Gly Thr Ser Lys Leu Val Gly ACX GGX ACX QRS AAM YTZ GTX GGX 817 Pro Ser Ser Asp lle Leu Asn lle CCX QRS QRS GAY ATH YTZ AAY ATH 841 Gln Gln Ala lle Gly Ala Thr Gln CAM CAM GCX ATH GGX GCX ACX CAM 865 Asn Gln Tyr Gly Glu Phe Asp lle AAY CAM TAY GGX GAM TTY GAY ATH 889 Asp Cys Asp Asn Leu Ser Tyr Met GAY TGY GAY AAY YTZ QRS TAY ATG 913 Pro Thr Val Val Phe Giu lle Asn CCX ACX GTX GTX TTY GAM ATH AAY 937 Gly Lys Met Tyr Pro Leu Thr Pro GGX AAM ATG TAY CCX YTZ ACX CCX 961 Ser Ala Tyr Thr Ser Gln Asp Gln QRS GCX TAY ACX QRS CAM GAY CAM 985 Gly Phe Cys Thr Ser Gly Phe Gln GGX TTY TGY ACX QRS GGX TTY CAM 1009 Ser Glu Asn His Ser Gln Lys Trp QRS GAM AAY CAY QRS CAM AAM TGG 1033 lle Leu Gly Asp Val Phe lle Arg ATH YTZ GGX GAY GTX TTY ATH LGN 1057 Glu Tyr Tyr Ser Val Phe Asp Arg GAM TAY TAY QRS GTX TTY GAY LGN 1081 Ala Asn Asn Leu Val Gly Leu Ala GCX AAY AAY YTZ GTX GGX YTZ GCX 1105 Lys Ala lle AAM GCX ATH 1129 wherein the 5' to 3' strand, beginning with the amine terminus and the amino acids for which each triplet codes are shown, and wherein abbreviations have the meanings defined in claim 3.

5. The bovine calf chymosin gene of claim 3, comprising the following deoxyribonucleotide sequence: Gly Glu Val Ala Ser Val Pro Leu GGG GAG GTG GCC AGC GTG CCC CTG 169 Thr Asn Tyr Leu Asp Ser Gln Tyr ACC AAC TAC CTA GAT AGT CAG TAC 193 Phe Gly Lys lle Tyr Leu Gly Thr TTT GGG AAG ATC TAC CTC GGG ACC 217 Pro Pro Gln Glu Phe Thr Val Leu CCG CCC CAG GAG TTC ACC GTG CTG 241 Phe Asp Thr Gly Ser Ser Asp Phe TTT GAC ACT GGC TCC TCT GAC TTC 265 Trp Val Pro Ser lle Tyr Cys Lys TGG GTA CCC TCA ATC TAC TGC AAG 289 Ser Asn Ala Cys Lys Asn His Gln AGC AAT GCC TGC AAA AAC CAC CAG 313 Arg Phe Asp Pro Arg Lys Ser Ser CGC TTC GAC CCG AGA AAG TCG TCC 337 Thr Phe Gln Asn Leu Gly Lys Pro ACC TTC CAG AAC CTG GGC AAG CCC 361 Leu Ser lle His Tyr Gly Thr Gly CTG TCT ATC CAC TAC GGG ACA GGC 385 Ser Met Gln Gly lle Leu Gly Tyr AGC ATG CAG GGC ATC CTG GGC TAT 409 Asp Thr Val Thr Val Ser Asn lle GAC ACC GTC ACT GTC TCC AAC ATT 433 Val Asp lle Gln Gln Thr Val Gly GTG GAC ATC CAG CAG ACA GTA GGC 457 Leu Ser Thr Gln Glu Pro Gly Asp CTG AGC ACC CAG GAG CCC GGG GAC 481 Val Phe Thr Tyr Ala Glu Phe Asp GTC TTC ACC TAT GCN GAA TTC GAC 505 Gly lle Leu Gly Met Ala Tyr Pro GGG ATC CTG GGG ATG GCC TAC CCC 529 Ser Leu Ala Ser Glu Tyr Ser lIe TCG CTC GCC TCA GAG TAC TCG ATA 553 Pro Val Phe Asp Asn Met Met Asn CCC GTG TTT GAC AAC ATG ATG AAC 577 Arg His Leu Val Ala Gln Asp Leu AGG CAC CTG GTG GCC CAA GAC CTG 601 Phe Ser Val Tyr Met Asp Arg Asn TTC TCG GTT TAC ATG GAC AGG AAT 625 Gly Gln Glu Ser Met Leu Thr Leu GGC CAG GAG AGC ATG CTC ACG CTG 649 Gly Ala lle Asp Pro Ser Tyr Tyr GGG GCC ATC GAC CCG TCC TAC TAC 673 Thr Gly Ser Leu His Trp Val Pro ACA GGG TCC CTG CAC TGG GTG CCC 697 Val Thr Val Gln Gln Tyr Trp Gln GTG ACA GTG CAG CAG TAC TGG CAG 721.

Phe Thr Val Asp Ser Val Thr lle TTC ACT GTG GAC AGT GTC ACC ATC 745 Ser Gly Val Val Val Ala Cys Glu AGC GGT GTG GTT GTG GCC TGT GAG 769 Gly Gly Cys Gln Ala lle Leu Asp GGT GGC TGT CAG GCC ATC CTG GAC 793 Thr Gly Thr Ser Lys Leu Val Gly ACG GGC ACC TCC AAG CTG GTC GGG 817 Pro Ser Ser Asp lle Leu Asn lle CCC AGC AGC GAC ATC CTC AAC ATC 841 Gln Gln Ala lle Gly Ala Thr Gln CAG CAG GCC ATT GGA GCC ACA CAG 865 Asn Gln Tyr Gly Glu Phe Asp lle AAC CAG TAC GGT GAG TTT GAC ATC 889 Asp Cys Asp Asn Leu Ser Tyr Met GAC TGC GAC AAC CTG AGC TAC ATG 913 Pro Thr Val Val Phe Glu lle Asn CCC ACT GTG GTC TTC GAG ATC AAT 937 Gly Lys Met Tyr Pro Leu Thr Pro GGC AAA ATG TAC CCA CTG ACC CCC 961 Ser Ala Tyr Thr Ser Gln Asp Gln TCC GCC TAT ACC AGC CAA GAC CAG 985 Gly Phe Cys Thr Ser Gly Phe Gln GGC TTC TGT ACC AGT GGC TTC CAG 1009 Ser Glu Asn His Ser t Gln Lys Trp AGC GAA AAT CAT TCC CAA AAA TGG 1033 lle Leu Gly Asp Val Phe lle Arg ATC CTG GGG GAT GTT TTC ATC CGA 1057 Glu Tyr Tyr Ser Val Phe Asp Arg GAG TAT TAC AGC GTC TTT GAC AGG 1081 Ala Asn Asn Leu Val Gly Leu Ala GCC AAC AAC CTC GTG GGG CTG GCC 1105 Lys Ala lle AAA GCC ATC 1129 wherein the 5' to 3' strand, beginning with the amine terminus and the amino acids for which each triplet codes are shown, and wherein abbreviations have the meanings defined in claim 3.

6. The bovine calf prochymosin gene of claim 4, comprising the following deoxyribonucleotide sequence Ala Glu GCT GAG 43 lle Thr Arg lle Pro Leu Tyr Lys ATC ACC AGG ATC CCT CTG TAC AAA 49 Gly Lys Ser Leu Arg Lys Ala Leu GGC AAG TCT CTG AGG AAG GCG CTG 73 Lys Glu His Gly Leu Leu Glu Asp AAG GAG CAT GGG CTT CTG GAG GAC 97 Phe Leu Gln Lys Gln Gln Tyr Gly TTC CTG CAG AAA CAG CAG TAT GGC 121 lle Ser Ser Lys Tyr Ser Gly Phe ATC AGC AGC AAG TAC TCC GGC TTC 145 Gly Glu Val Ala Ser Val Pro Leu GGG GAG GTG GCC AGC GTG CCC CTG 169 Thr Asn Tyr Leu Asp Ser Gln Tyr ACC AAC TAC CTA GAT AGT CAG TAC 193 Phe Gly Lys lle Tyr Leu Gly Thr TTT GGG AAG ATC TAC CTC GGG ACC 217 Pro Pro Gln Glu Phe Thr Val Leu CCG CCC CAG GAG TTC ACC GTG CTG 241 Phe Asp Thr - Gly Ser Ser Asp Phe TTT GAC ACT GGC TCC TCT GAC TTC 265 Trp Val Pro Ser lle Tyr Cys Lys TGG GTA CCC TCA ATC TAC TGC AAG 289 Ser Asn Ala Cys Lys Asn His Gln AGC AAT GCC TGC AAA AAC CAC CAG 313 Arg Phe Asp Pro Arg Lys Ser Ser CGC TTC GAC CCG AGA AAG TCG TCC 337 Thr Phe Gln Asn Leu Gly Lys Pro ACC TTC CAG AAC CTG GGC AAG CCC 361 Leu Ser lie His Tyr Gly Thr Gly CTG TCT ATC CAC TAC GGG ACA GGC 385 Ser Met Gln Gly lle Leu Gly Tyr AGC ATG CAG GGC ATC CTG GGC TAT 409 Asp Thr Val Thr Val Ser Asn lle GAC ACC GTC ACT GTC TCC AAC ATT 433 Val Asp lle Gln Gln Thr Val Gly GTG GAC ATC CAG CAG ACA GTA GGC 457 Leu Ser Thr Gln Glu Pro Gly Asp CTG AGC ACC CAG GAG CCC GGG GAC 481 Val Phe Thr Tyr Ala Glu Phe Asp GTC TTC ACC TAT GCN GAA TTC GAC 505 Gly lle Leu Gly Met Ala Tyr Pro GGG ATC CTG GGG ATG GCC TAC CCC 529 Ser Leu Ala Ser Glu Tyr Ser lle TCG CTC GCC TCA GAG TAC TCG ATA 553 Pro Val Phe Asp Asn Met Met Asn CCC GTG TTT GAC AAC ATG ATG AAC 577 Arg His Leu Val Ala Gln Asp Leu AGG CAC CTG GTG GCC CAA GAC CTG 601 Phe Ser Val Tyr Met Asp Ars Asn TTC TCG GTT TAC ATG GAC AGG AAT 625 Gly Gln Glu Ser Met Leu Thr Leu GGC CAG GAG AGC ATG CTC ACG CTG 649 Gly Ala lle Asp Pro Ser Tyr Tyr GGG GCC ATC GAC CCG TCC TAC TAC 673 Thr Gly Ser Leu His Trp Val Pro ACA GGG TCC CTG CAC TGG GTG CCC 697 Val Thr Val Gln Gln Tyr Trp Gln GTG ACA GTG CAG CAG TAC TGG CAG 721 Phe Thr Val Asp Ser Val Thr lle TTC ACT GTG GAC AGT GTC ACC ATC 745 Ser Gly Val Val Val Ala Cys Glu AGC GGT GTG GTT GTG GCC TGT GAG 769 Gly Gly Cys Gln Ala lle Leu Asp GGT GGC TGT CAG GCC ATC CTG GAC 793 Thr Gly Thr Ser Lys Leu Val Gly ACG GGC ACC TCC AAG CTG GTC GGG 817 Pro Ser Ser Asp lle Leu Asn lle CCC AGC AGC GAC ATC CTC AAC ATC 841 Gln Gln Ala lle Gly Ala Thr Gln CAG CAG GCC ATT GGA GCC ACA CAG 865 Asn Gln Tyr Gly Glu Phe Asp lle AAC CAG TAC GGT GAG TTT GAC ATC 889' Asp Cys Asp Asn Leu Ser Tyr Met GAC TGC GAC AAC CTG AGC TAC ATG 913 Pro Thr Val Val Phe Glu lle Asn CCC ACT GTG GTC TTC GAG ATC AAT 937 Gly Lys Met Tyr Pro Leu Thr Pro GGC AAA ATG TAC CCA CTG ACC CCC 961 Ser Ala Tyr Thr Ser Gln Asp Gln TCC GCC TAT ACC AGC CAA GAC CAG 985 Gly Phe Cys Thr Ser Gly Phe Gln GGC TTC TGT ACC AGT GGC TTC CAG 1009 Ser Glu Asn His Ser Gln Lys Trp AGC GAA AAT CAT TCC CAA AAA TGG 1033 lle Leu Gly Asp Val Phe lle Arg ATC CTG GGG GAT GTT TTC ATC CGA 1057 Glu Tyr Tyr Ser Val Phe Asp Arg GAG TAT TAC AGC GTC TTT GAC AGG 1081 Ala Asn Asn Leu Val Gly Leu Ala GCC AAC AAC CTC GTG GGG CTG GCC 1105 Lys Ala lle AAA GCC ATC 1129 wherein the 5' to 3' strand, beginning with the amine terminus and the amino acids for which each triplet codes are shown, and wherein abbreviations have the meanings defined in claim 3.

7. The bovine calf chymosin gene of claim 1, comprising the following deoxyribonucleotide sequence: Cys Leu Val Val Leu Leu Ala Val TGT CTC GTG GTG CTA CTT GCT GTC 1 Phe Ala Leu Ser Gln Gly Ala Glu TTC GCT CTC TCC CAG GGC GCT GAG 25 lle Thr Arg lle Pro Leu Tyr? Lys ATC ACC AGG ATC CCT CTG TAC AAA 49 Gly Lys Ser Leu Arg Lys Ala Leu GGC AAG TCT CTG AGG AAG GCG CTG 73 Lys Glu His Gly Leu Leu Glu Asp AAG GAG CAT GGG CTT CTG GAG GAC 97 Phe Leu Gln Lys Gln Gln Tyr Gly TTC CTG CAG AAA CAG CAG TAT GGC 121 lle Ser Ser Lys Tyr Ser Gly? Phe ATC AGC AGC AAG TAC TCC GGC TTC 145 Gly Glu Val Ala Ser Val Pro Leu GGG GAG GTG GCC AGC GTG CCC CTG 169 Thr Asn Tyr Leu Asp Ser Gln Tyr ACC AAC TAC CTA GAT AGT CAG TAC 193 Phe Gly Lys lle Tyr Leu Gly Thr TTT GGG AAG ATC TAC CTC GGG ACC 217 Pro Pro Gln Glu Phe Thr Val Leu CCG CCC CAG GAG TTC ACC GTG CTG 241 Phe Asp Thr Gly Ser Ser Asp Phe TTT GAC ACT GGC TCC TCT GAC TTC 265 Trp Val Pro Ser lle Tyr Cys Lys TGG GTA CCC TCA ATC TAC TGC AAG 289 Ser Asn Ala Cys Lys Asn His Gln AGC AAT GCC TGC AAA AAC CAC CAG 313 Arg Phe Asp Pro Arg Lys Ser Ser CGC TTC GAC CCG AGA AAG TCG TCC 337 Thr Phe Gln Asn Leu Gly Lys Pro ACC TTC CAG AAC CTG GGC AAG CCC 361 Leu Ser lle His Tyr Gly Thr Gly CTG TCT ATC CAC TAC GGG ACA GGC 385 Ser Met Gln Gly lle Leu Gly Tyr AGC ATG CAG GGC ATC CTG GGC TAT 409 Asp Thr Val Thr Val Ser Asn lle GAC ACC GTC ACT GTC TCC AAC ATT 433 Val Asp lle Gln Gln Thr Val Gly GTG GAC ATC CAG CAG ACA GTA GGC 457 Leu Ser Thr Gln Glu Pro Gly Asp CTG AGC ACC CAG GAG CCC GGG GAC 481 Val Phe Thr Tyr Ala Glu Phe Asp GTC TTC ACC TAT GCN GAA TTC GAC 505 Gly lle Leu Gly Met Ala Tyr Pro GGG ATC CTG GGG ATG GCC TAC CCC 529 Ser Leu Ala Ser Glu Tyr Ser lle TCG CTC GCC TCA GAG TAC TCG ATA 553 Pro Val Phe Asp Asn Met Met Asn CCC GTG TTT GAC AAC ATG ATG AAC 577 Ars His Leu Val Ala Gln Asp Leu AGG CAC CTG GTG GCC CAA GAC CTG 601 Phe Ser Val Tyr Met Asp Arg Asn TTC TCG GTT TAC ATG GAC AGG AAT 625 Gly Gln Glu Ser Met Leu Thr Leu GGC CAG GAG AGC ATG CTC ACG CTG 649 Gly Ala lle Asp Pro Ser Tyr Tyr GGG GCC ATC GAC CCG TCC TAC TAC 673 Thr Gly Ser Leu His Trp Val Pro ACA GGG TCC CTG CAC TGG GTG CCC 697 Val Thr Val Gln Gln Tyr Trp Gln GTG ACA GTG CAG CAG TAC TGG CAG 721 Phe Thr Val Asp Ser Val Thr lle TTC ACT GTG GAC AGT GTC ACC ATC 745 Ser Gly Val Val Val Ala Cys Glu AGC GGT GTG GTT GTG GCC TGT GAG 769 Gly Gly Cys Gln Ala lle Leu Asp GGT GGC TGT CAG GCC ATC CTG GAC 793 Thr Gly Thr Ser Lys Leu Val Gly ACG GGC ACC TCC AAG CTG GTC GGG 817 Pro Ser Ser Asp lle Leu Asn lle CCC AGC AGC GAC ATC CTC AAC ATC 841 Gln Gln Ala lle Gly Ala Thr Gln CAG CAG GCC ATT GGA GCC ACA CAG 865 Asn Gln Tyr Gly Glu Phe Asp lle AAC CAG TAC GGT GAG TTT GAC ATC 889 Asp Cys Asp Asn Leu Ser Tyr Met GAC TGC GAC AAC CTG AGC TAC ATG 913 Pro Thr Val Val Phe Glu lle Asn CCC ACT GTG GTC TTC GAG ATC AAT 937 Gly Lys Met Tyr Pro Leu Thr Pro GGC AAA ATG TAC CCA CTG ACC CCC 961 Ser Ala Tyr Thr Ser Gln Asp Gln TCC GCC TAT ACC AGC CAA GAC CAG 985 Gly Phe Cys Thr Ser Gly Phe Gln GGC TTC TGT ACC AGT GGC TTC CAG 1009 Ser Glu Asn His Ser Gln Lys Trp AGC GAA AAT CAT TCC CAA AAA TGG 1033 lle Leu Gly Asp Val Phe lle Arg ATC CTG GGG GAT GTT TTC ATC CGA 1057 Glu Tyr Tyr Ser Val Phe Asp Arg GAG TAT TAC AGC GTC TTT GAC AGG 1081 Ala Asn Asn Leu Val Gly Leu Ala GCC AAC AAC CTC GTG GGG CTG GCC 1105 Lys Ala lle AAA GCC ATC TGA TCA CAT CGC TGA 1129 CCA AGA ACC TCA CTG TCC CCA CAC 1153 ACC TGC ACA CAC ACA TGC ACA CAT 1177 GTA CAT GAG CAC ATG TGC ACA CAC 1201 ACA GAT GAG GTT TCC AGA CAG ATG 1225 ATT CTC AAT AAA CGT TGT CTT TCT 1249 GCA AAA AAA AAA AAA AA 1273 wherein the 5' to 3' strand, beginning with the amine terminus and the amino acids for which each triplet codes are shown, and wherein abbreviations have the meanings defined in claim 3.

8. A plasmid having the capability of replication in a procaryotic organism, comprising a deoxynucleotide sequence coding for bovine calf chymosin.

9. A plasmid having the capability of replication in a procaryotic organisms, comprising the bovine calf chymosin gene of claim 1, 2, 3, 4, 5, 6 or 7.

10. The plasmid of claim 9, wherein said procaryotic organism is of the genus Escherichia.

11. A microorganism transformed by the plasmid of claim 9.

1 2. The microorganism of claim 11, of the genus Escherichia.

13. The microorganism of claim 12, of the species coli.

14. A method for producing bovine calf prochymosin which comprises cultivating on an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals and growth factors, under bovine calf prochymosin-producing conditions, a procaryotic organism transformed by a plasmid capable of replicating in said organism and having a deoxynucleotide sequence coding for bovine calf prochymosin; and recovering bovine calf prochymosin so produced.

1 5. A method for producing bovine calf chymosin which comprises cultivating on an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals and growth factors, under bovine calf chymosin-producing conditions, a procaryotic organism transformed by a plasmid capable of replicating in said organism and having a doxnucleotide sequence coding for bovine calf prochymosin; and recovering bovine calf chymosin so produced.

1 6. The method of claim 14, wherein the procaryotic organism is Escherichia coli.

17. The method of claim 15, wherein said procaryotic organism is substantially similar to NRRL No. B-15061.

18. A biological pure culture of E. coli strain pGx1 225 NRRL No. B-15061.

19. A cloned bovine calf chymosin gene as claimed in claim 1 , substantially as hereinbefore described.

20. A method as claimed in claims 14 or 1 5 substantially as hereinbefore described.