FR2529570A1 - CHYMOSIN OR ISOLATED VEAL PROCHYMOSIN GENE AND USE THEREOF IN A PROCESS FOR THE PRODUCTION OF PRACTICALLY PURE CHYMOSIN OR PROCHYMOSIN OF VEAL - Google Patents

Abstract

THE INVENTION CONCERNS CLONED GENES OF CHYMOSIN AND PROCHYMOSIN OF CALF, PLASMIDS CONTAINING SUCH CLONED GENES, AND MICROORGANISMS TRANSFORMED BY THESE PLASMIDS. TO OBTAIN PRACTICALLY PURE CALF PROCHYMOSIN, IT IS GROWN ON AN AQUEOUS NUTRITIONAL MEDIA CONTAINING SIMILAR SOURCES OF CARBON, NITROGEN AND ESSENTIAL MINERALS, AND GROWTH FACTORS, IN CONDITIONS SUITABLE TO PRODUCE VEALYMOSIN , A PROCARYOTE ORGANISM TRANSFORMED BY A PLASMID CAPABLE OF REPLICATION IN ITS ORGANISM AND HAVING A SEQUENCE OF DEOXYNUCLEOTIDES CODING FOR CALF PROCHYMOSIN, THEN WE COLLECT THE CALF PROCHYMOSIN THUS PRODUCED. THE PROCARYOTE ORGANISM MAY BE ESCHERICHIA COLI.

Description

FIELD OF THE INVENTION The present invention relates to a bovine gene

cloned

  which specifies the biosynthesis of calf chymosin.

  The invention further relates to a plasmid containing a cloned calf chymosin gene and a microorganism

  transformed using 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 the pre-existing species

  Unweaned ruminants 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 specific elimination of 42 N-terminal amino acids. by a pH-dependent autocatalytic reaction. Calf chymosin exists as two isoenzymes (referred to as A and B) whose

  resolution can be performed by chromatography on DEAE-

  Crystalline enzyme cellulose Isoenzyme B, which may be catalytically less effective than isoenzyme A, is

  also the most abundant form in the tissue extracts.

  The isoenzyme B of calf chymosin was sequenced, and isoenzyme A was partially sequenced, revealing only one difference in amino acid level. from the rest 290 (the

  glycine in isoenzyme B and aspartic acid in iso-

  enzyme A) (see Foltmann, B et al, J Biol Chem, 254: 8447-

8456 l 1976 l).

  Calf chymosin (mixture of isoenzymes) was

  used for centuries for making cheese.

  Cepehdant, since the Second World War there has been a shortage of calf chymosin, due largely to a decline in the consumption of veal. As a result, chymosin obtained from the abomasal tissue (abomasum) of calves non-weaned has been regularly replaced by other animal and microbial enzymes, but none of them has the optimum characteristics

calf enzyme.

  As a result of this shortage, there has been considerable interest in bacterial cloning of a calf chymosin gene and in the development of a chymosin-producing microorganism. Various researchers have reported the extraction of m RNA of chymosin from

  Abomasal tissue of cattle (see H Uchiyama et al, Agric.

  Biol Chem, 44, 1373-1381 l 1980 l), and the cloning of a gene and chynmosin of double-stranded DNA (ds-c DNA), in E coli (see Beppu, T., et al., Recombinant DNA Industrial Applications (The Cetus Symposium) IFS Meeting, p. 21 (L) 1980 (1) Nishimori, K. et al. (J Biochem, 90, 901-904, 19811, describe attempts to clone a prochymosin in E coli and report a clone containing a 1021 base pair addition that apparently has sequential homology to the bovine chymosin gene (based on hybrid stopped translation observations). base pairs represents about 80% of the total length of the chymosin gene, assuming that the entire segment derives from the chymosin gene However, the authors report no characterization data for the gene, for example sequential analysis information or a factorial restriction map, and therefore we do not know The extent to which the cloned fragment derives from the calf chymosin gene. Moreover, the authors do not report the observation of any expression of the proteins by

their bacterial clones.

  There is therefore a continuing need for a whole calf and cloned chymosin gene, as well as a strain.

  microbial capable of producing calf chymosin.

  The present invention relates to an intact and isolated calf chymosin gene, an intact and isolated calf prochymosin gene, plasmids containing such genes, and microorganisms transformed with such plasmids. production of veal chymosin, which consists in cultivating a microorganism producing veal chymosin on a nutrient medium under conditions

  to produce chymosin.

  The procedures which make it possible to obtain and clone a calf chymosin gene and which are described herein are, unless otherwise indicated, carried out using standard techniques of molecular biology. See for example Ullrich, A et al, Science , 196, 1313 (1977) and Seeburg, PH et al., Nature, 270, 486

(1977).

  Procedures leading to a micro-

  The calf chymosin-producing organism can be divided into the following major steps, each of which is described more fully here: (1) extraction of prochymosin m RNA from the milk-fed calf-coating skin mucosa, (2) ) in vitro synthesis of double-stranded DNA, using the mRNA of calf prochymosin as "template", (3) insertion of the double-stranded DNA into an appropriate cloning vector and transformation of the cells with this cloning vector, and 4) Selection of microbial clones containing the calf prochymosin gene from their ability to express calf prochymosin The experimental details of these techniques are set out below in the "EXAMPLES" section, but it goes without saying that, to the

  light of the present description, it is possible to modify

  these techniques in an important way without stopping

to achieve the desired results.

  The mRNA of bovine prochymosin is advantageous

  recovered in the form of po-lysomal mRNA from the crushed coating mucosa of milk-fed calves Ribonucleases are present in this tissue at relatively high concentrations, so the tissue is preserved by freezing it shortly after the death of the animal, and are there used during the extraction operations substances which reduce the activity of ribonucleases In general, the procedure of C Vaslet et al, Nature, 285, 674676 ( 1980) can be used to extract the mRNA-polysomal The frozen tissue can be pulverized and homogenized and, after preliminary centrifugation to remove lipids and insoluble cell debris, it is advantageous to separate the relatively dense polysomal m m, by centrifugation in a sucrose gradient the m polyadenylated RNA may conveniently be recovered from the preparation of m polysomal RNA by elution on a column of oligo (T) - cell ulose (see Green, M et al., Arch Biochem Biophys,

172, 74 (1976).

  To obtain a preparation enriched with prochymosinc m RNA, the oligo d (T) -cellulose column eluate can be fractionated by sucrose gradient centrifugation, according to the well-known technique. prochymosin RNA of m RNA fractions obtained from centrifugation in the sucrose gradient, by determining their translational activity in a translation system without cells A number of cell-free translation systems have been devised, such as wheat germ extract (Martial, J et al, Proc Nat'l Acad Sci USA, 74, 1816 (1977)), an mRNA-dependent retriculocyte lysate (Pelham, HRB et al, Eur J Biochem, 67, 247 (1976)), and Xenopus laevis oocytes (Sloma, A et al, Methods in Enzymology, Vol 70 (1981)). The system based on wheat germ extract is preferred for the analysis of m Prochymosin RNA The wheat germ system lacking cell can be supplemented with a radioactively labeled amino acid, such as 535-methionine, for the resulting proteins to contain a label. It is then convenient to analyze the prochymosin content of the various products of the gel-electrophoresis gel, followed by a visualization by fluorography, and one uses for the synthesis of the ds-c DNA the fraction of the m RNA which was revealed

  produce prochymosin in this system.

  This enzyme catalyzes the synthesis of a single strand of AD1 from deoxynucleoside triphosphates on the m RNA template (see Kacian, DL, et al. , Proc. National Acad Sci USA, 73, 2191 (1976) The poly r (A) tail of the m RNA makes it possible to use

Z 529570

  oligo (dT) (from about 12 to 18 nucleotides) as a primer for the synthesis of the c DNA The use of a radioactively labeled deoxynucleoside triphosphate facilitates the control of the synthesis reaction Generally, it is possible to it is advantageous to use a phosphorus-containing deoxynucleoside triphosphate 32, for example 32 pd CTP. Synthesis of the c DNA is generally carried out by incubating a solution of the mRNA, the triphosphates of

  deoxynucleoside, oligo (d T) and reverse transcriptase.

  The solution also preferably contains small amounts of actinomycin D and dithiothreitol to promote full synthesis (see Kacian, DL, et al., Supra). After incubation, ethylenediaminetetraacetic acid (EDTA) is added to the solution, and the solution is extracted with a mixture of phenol and chloroform. It is advantageous to purify the aqueous phase by gel filtration chromatography, and the complex is precipitated with alcohol.

c mDNA mRNA of the eluate.

  M RNA can be selectively hydrolyzed in the presence of cDNA with sodium hydroxide diluted at elevated temperature. Neutralization of the alkaline solution and precipitation with alcohol provide a copy of the single-stranded DNA.

  It has been shown that the copy of the mono-

  catenary had a 5 '-poly (dT) tail, and had a

  3 '-terminal hairpin structure, which provides a

  Short duplex DNA segment (Efstratiadis, A, et al., Cell, 7, 279 (1976). This 3 'hairpin structure can act as a primer for the synthesis of a second strand of DNA. leads the synthesis of this second strand substantially under the same conditions as the synthesis of the copy of the c DNA, except that the reverse transcriptase is replaced by the Klenow fragment of the E coli DNA polymerase I (Klenow, H, et al., Eur J Biochem, 22, 371 (1971)) The c duplex DNA that is recovered by this procedure has a 3 'loop, resulting from the 3' hairpin structure of the copy of the c. Single-stranded DNA This 3 'loop can be cleaved by digestion with an enzyme, Si nuclease, using substantially the procedure of Ullrich, A, et al, supra. The result of digestion with Si nuclease can be extracted with a mixture of phenol and chloroform, and the ds-c DNA resulting from the

aqueous with alcohol.

  For development and selection purposes, the ds-c DNA gene, prepared as described above, is generally introduced into a suitable cloning vector, which

  it is used to transform the appropriate host cells.

  Suitable cloning vectors include various plasmids and bacteriophages, but plasmids are generally preferred. The criteria for selection of a cloning vector include its size, replicability in host cells, the presence of selectable genes, and the presence of cloning vectors. a site for insertion of the gene as for its dimensions, it is advantageous that the vector is relatively small to allow insertion of large genes, and so as not to divert large amounts of nutrients 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 number of copies per cell The genes specified One or more phenotypic properties, preferably antibiotic resistance, facilitate the selection of the transforming principles. The insertion site is advantageously a unique restriction site for a restriction endonuclease. A cloning vector meeting all of these criteria is plasmid p BR 322, Bolivar, F., et al Gene, 2, 95 (1977) This plasmid is small (approximately 2.8 x 10 6 daltons), carries ampicillin resistance genes (amp) and tetracycline (tet), and is subject to relaxed replication in E coli The plasmid also has a unique restriction site for the endonuclease, Pst I, which appears within the "amp" gene. The ds-c DNA can conveniently to be inserted into this plasmid by a "queuing" homopolymer technique (Nelson, T, et al, Methods of Enzymology, 68, 41 (1980) Homopolymer tails, for example poly-d C, are added to the 3 of the ds-c DNA, by reaction with the suitable deoxynucleoside triphosphate, eg CTP, in the presence of

  terminal deoxynucleotidyl (Chang, L M S, et al., J. Biol.

  Chem, 246, 909 (1971)) ds-c DNAs with poly (C) tails

  are preferably sized on gradients of -

  neutral sucrose (see Norgard et al., J Biol Chem, 255, 7665-7672 (1980)). Use in subsequent steps

  double-stranded DNA longer than 600 base pairs.

  The plasmid is opened by digestion with endocrine

  appropriate nuclease, and tails of complementary homopolymers, for example poly d G, are added to the 3 '-hydroxyls of the open plasmid using the same homopolymer quenching technique, using the GTP d for example. Instead, queuing reactions can be monitored by

  using triphosphates of radioactive deoxynucleotide

  In general, the reactions are carried out in such a way as to obtain tails which are approximately 10 to 20 nucleotides in length. The cDNAs are recovered. Plasmid, for example by phenol extraction followed by an alcohol precipitation. The two "tailed" DNA species are "annealed" by incubating a buffered solution of equimolar concentrations of the two species, so as to obtain a plasmid. recombinant containing the calf prochymosin gene. It is possible to transform an appropriate amps strain of E. coli with the recombinant plasmid into

  substantially using the method of Lederberg, J Bacterio-

  logy, 119, 1072 (1974) The transforming principles are usually grown on a normal L broth containing tetracycline. Colonial samples growing on the medium containing the

  tetracycline in a second medium containing ampicillin.

  Since the plasmid p BR 322 gives the cells a resistance to tetracycline, the colonies which grow on the medium containing the tetracycline must contain this plasmid. On the other hand, the ampicillin resistance of the plasmid p BR 322 is destroyed by the insertion. of the gene, therefore, only for subsequent analysis are

R S

  In general, from a few hundred to several thousand potential prochymosin clones are produced by these procedures. To identify a smaller group of colonies that contain the prochymosin gene, it may be advantageous to employ a radioactively labeled DNA probe (see Grunstein, M, et al, Proc Nat'l Acad Sci USA, 72, 3961-3965 (1975) Such a DNA probe can advantageously be prepared from a population of m RNA very enriched in prochymosin, using '

  reverse transcriptase and incorporating a nucleotide -

  Radiolabelled In a general way, 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 performing hybridization analysis of

colonies.

  Colony hybridization generally has the effect of reducing the number of potential prochymosin clones, but a considerable number of these clones usually remain. One procedure that has been successfully used to complete the sorting of these clones involves Enzyme-Linked Immunosorption ("ELISA") This assay detects chymosin in clones where protein expression is conserved. The assay involves the application of chymosin standards and cell lysates to the interior surfaces of cuvettes. a microtiter plate made of plastic material, and to react immunoogically

  the chymosin possibly present with the antiscrps

  Chymosin This hybridized conjugate is then reacted with a second enzyme-labeled antibody which is specific for the first antibody. After separating the unbound labeled antibody, a chromogenic substrate is introduced to determine the bound enzyme that is present, which causes is a measure of the chymosin content of the original cell lysate. This technique has proven very useful for isolating a few clones from a large population of clones identified as

  being positive by colony hybridization analysis.

  It is furthermore advantageous to characterize the few clones identified by the enzyme-linked immunosorption assay, by restriction factor map and by sequential analysis. One clone, designated by the reference p Gx 1225, consistently gave strongly positive results. ELISA and sequence analysis and restriction map of the factorial also confirmed that it contained the gene for prochymosin the factorial map of restriction to the addition of p prochymosin gene Gx 1225

  is shown in Figure 1 of the accompanying drawings.

  The addition consists of 1289 base pairs zymogen is specified by the region from base pair 43 to base pair 1135, and the mature chymosin gene is specified

by the base pairs 169 to 1137.

  The nucleotide sequence of this prochymosin gene addition was determined by the method of Sanger, et al Proc Nat'l Acad Sci USA, 74, 5463-5467 (1977), and this nucleotide sequence is as follows: D 1 he D v D aqa asv 1 D 1 DD 1 D 9 D 1 D v D v D jas -las i I 9 1 qj, C 3 S -'l

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  A A G C C A T T C T G AT C AT C AT CG T G A

  C CA A GA AC C T C A C T G T C CC C A C

  A C C T G C A C A C A C A C AT G C A C A C AT

  G T A CA T GA G C AC T T G T G C A C A C A C

  A C A G A T GA G G T T TCC A G A C A G AT G

  A TT CT C A A T A A C C T TGT C T T T C T

G C A AAA AAA AAA AA AA

  This figure represents the 5 '-> 3' strand of the non-coding and coding regions, as well as the amino acid sequence that is specified by the above gene and in

  the whole description, the abbreviations have the

  Commonly used meanings: A = deoxyadenyl T = thymidyl G = deoxyguanyl C = deoxycytosyl GLY = 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 HIS = histidine PRO = proline GLN = glutamine ASN = asparagine It will be appreciated that, due to the degeneration of the genetic code, the nucleotide sequence of the gene may To a large extent, for example, parts or the entire gene could be synthesized chemically to give the DNA having a nucleotide sequence different from that shown above, but where the amino acid sequence would be conserved. , provided that the appropriate codon-amino acid assignments are respected Having established the nucleotide sequence of the calf prochymosin gene and the amino acid sequence of the protein, the gene of the present invention is not limited to a particular nucleotide sequence, but includes all its authorized variants

by the genetic code.

  A culture of E. coli cells, p Gx 1225, containing the prochymosin gene addition described above, was deposited at the United States Department of Agriculture, Northern Regional Research Laboratory, Peoria, Illinois.

U.S A, under No. B-15061 of NRRL.

  The present invention has been described with respect to the use of E. coli as a bacterial host for recombinant DNA containing the calf prochymosin gene, but qualified biologists will realize that other gram-negative bacteria, such as Pseudomonas, Gram-positive bacteria, such as Bacillus, and higher single-cell organisms, such as yeasts and fungi, can be used for cloning and / or expression of the prochymosin gene or portions thereof.

of it.

  The invention will be further illustrated by the following examples, which should not be considered

as limiting.

EXAMPLE I

  Preparation of Polysomal RNA The milk-fed calves were collected at a local abattoir (Henry Stapf, Inc., Baltimore, Maryland) within half an hour after the animal was slaughtered. coating mucosa and rapidly frozen in liquid nitrogen for preservation Polysomal mRNA was prepared according to a variant of the procedure described by Vaslet et al (Nature 285: 674-676 f 1980) Sprayed frozen tissues (50 g) were homogenized in 100 ml buffer containing 0.25 M KCl, 0.025 M MgCl 2, 0.05 M tris-HCl at pH 7.4.4, 5% of

  NP 40, 200 μg / ml cyclohexamide, and 250 μg / ml heparin.

  The homogenate was centrifuged twice at 10,000 rpm for 10 minutes at 4 ° C to lighten a thick layer of lipids and remove cell debris. The polysomes were collected as a pellet prepared by centrifuging the supernatant. at low speed in a "cushion"

  2.5 ml of 52% sucrose in homogenization buffer

  which did not contain NP 40, cyclohexamide and heparin at 28,000 rpm for 8 hours at 4 ° C. in a Beckman SW 28 rotor. This polysomal pellet was resuspended in mM tris-HCl H 9, 1 mM EDTA, 150 mM acetate acetate

  sodium, 1% SDS containing proteinase K (250 μg / ml).

  After incubation for 60 minutes at 37 ° C., the preparation was extracted twice with a mixture of phenol and chloroform and the RNA was precipitated with alcohol at 0 ° to 80 ° C. for one hour. Pelleted RNA and washed three times with 3 M sodium acetate to solubilize small amounts of RNA, mucin and heparin. The final polysomal RNA pellets were resuspended in deionized water and we have

kept at 80 C.

  Poly (A) plus mRNA was obtained by

  hybridization of the polysomal RNA in a column of oligo d (T) -

  cellulose Ona equilibrated this column with a binding buffer containing 10 mM tris-HCl at pH 7.6, 1 mM EDTA, 0.1% O SDS, and 400 mM NaCl, and The whole of the polysomal RNA preparation was run five times in the column. The unbound RNA was eluted with a volume of binding buffer equal to several times the volume of the column. An elution buffer containing 10 mM tris-CiI at fold 7.6, 1 mM EDTA and 0.1% SDS to drive out of the column bound poly (A) and mRNA was precipitated from the column. alcohol on

  poly (A) and mRNA and were stored at 80 C.

EXAMPLE II

  Fractionation of poly (A) + m RNA in a sucrose gradient Poly (A) + of m RNA was decanted on a continuous gradient of 5% to 20% of sucrose density constituted in an SDS buffer containing 0, 5% SDS,

  0.1M NaCl, 0.01M tris HCl at pH 7.5, 0.001M EDTA.

  The RNA was heated at 80 ° C. for 2 minutes and cooled rapidly on ice just before being decanted on the gradient. Separate tubes were placed as sedimentation standards. Calf RNA-RNA The gradients were rotated by themselves at 30,000 rpm for 20 hours at 22 ° C. in a Beckman SW 40 rotor, and 0.6 ml fractions were collected to generate a profile. A 260 which is shown in FIG. 2 of the accompanying drawings. The mRNA of each fraction was rinsed with alcohol, and its translational activity was then analyzed in a commercial wheat germ system (BRL) preparation. ) (see Example III) The bovine hachurea fraction of Figure 2 is enriched in bovine prochymosin mRNA, and

  it was used for the synthesis of ds-c DNA (Example IV).

EXAMPLE III

  Translation on wheat germ of mRNAs and polyacrylate gel electrophoresis Translations of MRA samples into a cell-free wheat germ system, supplemented with S methionine, were carried out. suggested by the supplier (Bethesda Research Laboratory, Rockville, Maryland) The wheat germ products were electrophoresed in SDS-polyacrylamide plate gels containing a 5% stack and 12.5% separation gel ( Laemmli, Nature 227: 680-685 l 1970 l) Radiolabeled proteins were visualized by fluorography (Bonner and Laskey, Eur J Biochem 46: 83-88 1974 l with a Kodak film

XAR-5.

EXAMPLE IV

  Preparation of Hybrid Plasmids Double-stranded DNA and homopolymeric "queuing" synthesis reactions were performed with only slight modifications, as detailed in McCandliss et al. (Methods of Enzymology, Vol 70, 1981) About 20 G residues were added to the 3 'end of Pst I linearized p BR 322. The same number of C residues were added to the ds-c DNA synthesized on sized ds-c C-tailed DNA on neutral sucrose gradients as disclosed by Norgard et al. (JBC 255: 7665-7672 1980 1). ds-c DNA was dissolved in distilled water and was decanted on a cooled gradient of 12 ml of 5% -25% sucrose containing mM tris-HCl at pH 7.5 and 1 mM EDTA This gradient was rotated on itself at 38,000 rpm for 17.5 hours, at 5 ° C., in a Beckman SW 40 rotor. At the same time, a second gradient containing 10 μg of plasmid p BR 322 digested with Taq I endonuclease was used. Sizing Standard The gradients were fractionated and analyzed as follows: a) Taq digested gradient p 322. The DNA of each 0.5 ml fraction was precipitated with alcohol. it was resuspended in a sample buffer containing 0.09 M tris-HCl at pH 8.0, 0.09 M boric acid, 40 mM EDTA, 5% glycerol, 0.025 % xylene cyanol and 0.025% bromophenyl blue Each sample was electrophoresed on an 8% acrylamide plate gel. Ethidium bromide gel was photographed and lanes were noted. containing

  DNA larger than 600 base pairs b) gradient ds-

  100 μl of each 1 ml fraction were added to the scintillator for counting. The DNA of each fraction was precipitated with alcohol, and those which contained dsc DNA longer than the following were used in subsequent steps. 6 o O base pairs (based on information provided by

  the gel of p BR 322 digested with Taq I).

  Oligo d (G) -panted BR 322 DNA was diluted at equimolar concentrations and ds-c oligo d (C) sized sieved DNA in 10 mM tris-HCl to pH 7, 5, 100 m M

  NaCl and 2.5 mM Na 2 EDTA were annealed at 70 ° C.

  10 minutes and then cooled slowly

night at room temperature.

EXAMPLE V

  Transformation Recombinant plasmids were introduced into E coli HB 101, substantially according to the method of Lederberg and Cohen (J Bacti 119: 1072, 1974 l).

  transforming principles on standardized LB-plates

  tetracycline (15 μg / ml) at 37 ° C. for 24 hours. Colonies were collected on LB-ampicillin plates (50 μg / ml) to determine the phenotype. Only ampicillin sensitive colonies were selected for analysis.

  Subsequently, about 750 clones were isolated.

EXAMPLE VI

  Colony Hybridization Transformative principles for colony hybridization were prepared according to a modification of the procedure originally described by Grunstein and Hogness (PNAS, 72: 3961-3965, 1975 l). hybridization of DNA-bound filters in the presence of dextran sulfate and

  formamide, as suggested by Wahl et al (PNAS 76: 3683-

  3687 l 1979 l) The filters were pre-hybridized in 6x SSC (20X = 3M NaCl, 0.3M sodium citrate);

  1 x Denhardts solution (10 X 0.2% BSA, 0.2% PVP-

  ; 0.2% Ficoll); 0.1% SDS in a glass jar with

  gentle stirring at 42 C for 60 minutes

  Hybridization followed in a filter 8 mls of 50% formamide, 5 x SSC, 5 x Denhardt's solution, 50 mM NaPO 4 at pH 6.5, 1% glycine, 195 gg / ml of DNA denatured salmon sperm for 2 hours at approximately 42 ° C. with gentle stirring A 32-c DNA probe (= 0.5 × 10 6 cpm / filter) was added a DNA Pc probe (= 0.5 × 10 cpm / filter) to a hybridization mixture containing 50% formamide, 5 x SSC, 1 x Denhardt's solution, 20 mM NaPO 4 at pH 6.5, 10% dextran sulfate, 100 g / ml of denatured salmon sperm DNA, and incubated with the filters at 42 ° C. for 18 hours with gentle shaking. This probe was prepared from a sucrose gradient fraction highly enriched in prochymosin m RNA ( Example II) which was the fraction immediately higher than that used for the synthesis of ds-c DNA. The filters were washed three times for minutes in 2 x SSC, 0.1% SDS at room temperature and 2 x 10 minutes. ns 0.1 x SSC, 0.1% SDS at 50 C Positives were visualized by exposing the Kodak XR-5 film-stained filters overnight to -80 C with a display

enhancer.

EXAMPLE VII

  Enzyme Linked Immunosorbtion Assay The Enzyme Linked Immunosorbent Assay (ELISA), submitted by Engvall, E and Pearlman, P (Immunochemistry, 8: 871-874, 1971 l), was used to sort the 135 clones. recombinants which had previously been identified as positive by the procedure of Example VI Preparation of the antibody: the chymosin (Sigma) was repurified on an isoelectric focusing column, and the purified chymosin was used as immunogen in all subsequent injections New Zealand white rabbits were initially immunized with 1 mg of chymosin in a

  Freund's complete adjuvant administered intravenously

  The recalls contained 0.5 to 0.75 mg of chymosin in incomplete Freund's adjuvant and were administered similarly at fortnightly intervals for three months. Blood samples were then taken and

  used to prepare antichymosin serum.

  Analysis: Colony overnight cultures were placed in 5 ml of L-broth containing 25 mg / liter

  of tetracycline Negative control

  F 101 cultures containing plasmid p BR 322 The cultures were spun on themselves in a Sorval SM-24 rotor for 5 minutes at 4 ° C. and 8000 rpm. The cells were washed once in saline. phosphate buffered saline (PBS), were transferred to eppendorf tubes and resuspended in 300 ml of 20 mM carbonate-bicarbonate buffer at pH 9.6, and placed on The ice was treated with sonic waves on the ice for 10 to 15 seconds at 200 watts. The debris was pelleted and the lysate was cautiously transferred to a clean eppendorf tube. Cell lysate at each cuvette of a microtiter plate (Linbro, Flow Labs) Three families of standard curves were prepared with repurified chymosin.

  ranging from 1 gg / ml to 0.1 gg / ml in half dilutions.

  Each standard (200 g) was added to the appropriate cuvettes of the microtiter plate, which was incubated overnight at 4 ° C. The cell lysates and standards were aspirated and washed. three times with PBS

and 0.05% of "Tween 20".

  200 μl of 3% rabbit serum albumin was added to each cuvette and incubated at 37 ° C for 1 hour to block remaining active binding sites on the plastic. The plate was washed three times with sterile tissue. PBS-Tween ™, and 200 g 1 of antichymosin antibody (diluted 1/32) were added to each cuvette, except for one set of standards which received pre-immune serum diluted 1: 1. The plate was incubated at 37 ° C. for one hour and washed three times with "PBS-Tween". 200 μl of peroxidase-conjugated anti-rabbit antibody was added to each well. of horseradish (Sigma) and diluted to 1/500, and incubated for one hour at 37 ° C. After washing it three times with "PBS-Tween", it was added to each cuvette

  2,2'-azino-3-ethylbenzthiozolin

  sulfonatel (ABTS) The plate was read on a "Flow Multi-

  Scanning at 405 nm 15 minutes after substrate addition Two clones were identified by this procedure as giving consistently positive results. One of these clones, designated p Gx 1225, contained an addition of sufficient length to coding for a complete calf prochymosin gene The DNA of this clone was characterized by restriction factor map and sequential analysis, and a cell culture was deposited at the United States Department of Agriculture, Northern Regional Research.

  Laboratory, Peoria, Illinois as NRRL B-15061.

. "

Claims (15)

CLAIMS Isolated calf chymosin gene consisting of a cloned deoxyribonucleotide sequence, characterized in that said sequence codes for calf chymosin.   2 Isolated calf prochynsin gene consisting of a cloned deoxyribonucleotide sequence, characterized in that said sequence code for the calf prochymosin.   3. Calf chymosin gene according to claim 1, characterized in that it comprises the following deoxyribonucleotide sequence: Gly Glu Val Ala Ser Val Pro Leu   G G X G A M G T X G C X Q R S G T X C C X Y T Z Thr Asn Tire Leu Asp Asp Gln Tyr   ACX A Y Y Y Y Z Z A Y Q R S CA M TAY Phe Gly Lys Island Tyr Leu Gly Thr   TTY G G X AA M A T H T A Y YT Z G G X ACX Pro Gln Pro Gln Phe Thr Val Leu   CCX C C X CA M GA T Y AC X G T X Y T Z Asp Thr Phe Gly Ser Ser Asp Phe   T T Y G A Y A C X G G X Q R S Q R S G Y Y Y Trp Val Ser Ser Pro Tyr Cys Lys   TGG GT X C CXQ RS AT H T Y Y Y Y M Ser Asn Ala Cys Lily Asn His Gln   QRS AA Y G C X Y Y Y Y M A Y C A Y CA M Arg Phe Asp Pro Arg Lys Ser Ser   LGN TT Y G A Y CC Y LG N Y M QR S QRS Thr Phe Gln Asn Leu Gly Lys Pro   ACX T T Y Y A Y Y Y Z GG X AA M DC X Leu Ser Her Tyr Island Gly Thr Gly   YT Z Q R S AT H C A Y T Y G G X AC X G G X Ser Met Gln Gly Ile Leu Gly Tyr   QRS AT G CA GG M X Y Asp Thr Thr Val Val Val Asn Island   G A Y A C X G T X A C X G T X Q R S A Y Y T H Val Asp Gln Island Gln Thr Val Gly   GTX G A Y A T H CA M AC M X G TX GG X Leu Ser Thr Gln Glu Pro Gly Asp   YT Z Q R S AC X CA M GA M X X G G X GAY Val Phe Thr Ala Tyr Glu Phe Asp   GTX TT Y AC X TA Y G C X GA M TT Y GAY Ile Leu Gly Met Ala Tyr Pro   A T H Y T Z G G X A T G G C X T A Y C C X Ala Ser GCX QRS Phe Asp T T Y G A Y Leu Val YT Z GTX Val Tyr GTX TAY Glu Ser GAM QRS Asp Island AT EH GAY Ser Leu QRS YT Z Val Gln G T X C A M Val Asp GTX GAY Val Val GTX GTX Cys Gln TGY CAM Thr Ser ACX QRS Ser Asp QRS GAY Ala Island G C X ATH Tyr Gly TAY GGX Asp Asn GAY AAY Val Val G T X G T X Met Tyr A T G T A Y Glu Tyr G A M T A Y Asn Met A Y A T G Ala Gln G C X CAM Met Asp AT GAY GAY Met Leu AT G Y T Z Pro Ser C C X Q R S His Trp C A Y T G G Gln Tyr C A M T A Y Ser Val Q R S G T X Val Ala G T X G C X Ala Island G C X AT H Lys Leu A A M Y T Z lie Leu AT T H Y T Z Gly Ala G G X G C X Glu Phe G A M T T Y Leu Ser Y T Z Q R S Phe Glu T T Y G A M   Ser Q R S Met A T G Asp GAY Arg L G N Thr A C X Tyr T A Y Val G T X Trp T G G Thr A C X Cys T-G Y Leu Y T Z Val G T X Asn A A Y Thr A C X Asp GAY Tyr T A Y A T H H A T H Asn -A TO Y   Leu Y T Z Asn A A Y Leu YT Z Tyr T A Y -Pro C C X Gln C AM Ile A T H Glu G A M Asp - G A Y Gly G G X Ile A T H Gln C A M A T H H G A L A A Y Y Pro Leu Thr Pro C C X Y T Z C X C C X   Gly GGX Ser QRS Pro CC X Arg LGN Phe TTY Gly GGX Gly GGX Thr ACX Val GTX Phe TTY Ser QRS Gly GGX Thr ACX Pro CCX Gln CAM Asn AAY Asp GAY Pro CCX Gly GGX Leu YTZ Val 1 GTX His CAY Ser QRS Gln CA M Ala GCX Gly GGX Thr ACX Thr ACX Gly GGX Gly GGX Gly GGX Ser QRS Gi n CAY Gin CA M Cys TGY Thr ACX Lys AAM Ala Tyr Thr Ser Gln Asp G C X T A Y A C X Q RS C A M GAY Phe Cys Thr Ser Gly TTY TG Y AC X Q R S GG X Gln CA M Phe Gln T T Y CAM Glu Asn His Ser Gln Lys Trp GA M A Y Y CA Y Q RS CAM Y M M TG G Leu Gly Asp Val Phe II-e Arg   Y T Z G G X G A Y GT X T T Y A T H L G N Tyr Tire Ser Val Phe Asp TA Y T Y Y RS G T X TT Y GAY Asn Asn A Y A A Y Leu Val Arg L G N Gly Leu Ala Y T Z G T X G G X Y T Z G C X Ala Island G C X AT H   wherein strand 5 'to 3', beginning with the amino terminus, and the amino acids for which each triplet codes, are represented, and wherein, within each triplet, A is deoxyadenyl T is thymidyl G is deoxyguanyl -C is deoxycytosyl X is A, T, C or GY is T or C When Y is C, Z is When Y is T, Z is H is A, T or CQ is T or A When Q is T, R is When Q is A, R is M is A or G L-is A or C When L is A, N is When L is C, N is GLY is glycine A, T, C or GA or GC and S is A, T, C or GG and S is T or CA or GA, T, C or G Ser Q RS Gly GGX Ser QRS Ile AT H Glu GA M Ala GCX Lys AAM ALA is alanine VAL is valine LEU is leucine ILE is SER isoleucine is serine THR is threonine PHE is phenylalanine TYR is tyrosine TRP is tryptophan CYS is cysteine MET is ASP methionine is GLU aspartic acid is glutamic acid LYS is ARG lysine is arginine HIS is the histidi does PRO is proline GLN is glutamine ASN is asparagine.   4-calf prochymosin gene according to claim 2, characterized in that it comprises the following deoxyribonucleotide sequence: Ala Glu G A M Thr Arg A C X L G N Pro Island At T H C C X Leu Tyr Lys Y T Z T A Y A A M Ser Lys Leu Leu Arg Lys Ala Leu AA M Q R YT Y LG Y Y AA GC X YT Z Glu His Gly GAM C A Y GGX Leu Gln Lys YT Z CA M AA M Ser Ser Lys QRS Q R S AAM Leu Leu Glu Asp Y T Z Y T Z G A M GAY Gln Gln Tyr Gly C A M C A M T A Y G G X Tyr T A Y Ser Gly Q R S G G X Glu Val Ala Ser Val G A M G T X C X Q R S G T X Asn Tyr Leu Asp Ser A Y Y Y Z G Y Y Y Y R Gly Lys Ile Tyr Leu G G X A A M A T H T A Y Y T Z Phe T T Y Pro Leu C C X Y T Z Gln Tyr CAM T A Y Gly Thr G G X A C X   G C X Is A T H Gly G G X Lys A A M Phe T T Y Is A T H Gly G G X Thr AC X Phe TT Y Pro Pro Gln Glu Phe Thr Val C C XC CX C AM G AM T TY A CX GT X Phe Aspv Thr Ser Gly Ser Asp T TY GA Y -A CX G G Q Q Q Q RS GA Y Trp Pro Val Ser Ser Tyr Cys TGG GGT-X C CX Q RS AT TH T AY T GY Ser Asn Ala Cys Lys Asn His Q R SAA Y G CX T GY AM A AY CA Y Arg Phe Asp Pro Arg Lys Ser L G NT TY G AY C C L L A A AM QR S Thr Phe G In Asn Leu Gly Lys C X T T Y C AM A Y Y TZ G GX AA M Leu Ser Her Tyr Gly Thr Island T Z Q RS AT TH C AY T AY G GX AC X Ser Met Gln Gly Ile Leu Gly RS TO TG C AMG GX TO TH Y T Z GG X Asp Thr Thr Val Val Ser Asn AY TO CX G TX TO CX G TX Q RS AA Y l 33 Val Asp Gln Gin Thr Island Val Val T X G AY A TH C AM C AN A CX GT X Leu Ser Thr Gin Glu Pro Gly TZ Q RS A CX C A M G AN C CX G GX Val Phe Thr Tire Ala Glu Phe T X TTY A CX AY G CX G AN T TY Giy Island Leu Gly Met Ala Tyr G XA TH Y T Z G GX A TG G CX T AY Ser Leu Ser Glu Ser Ser Ser R SY TZ G CXQ RS G AM T AY QRS Pro Val Phe Asn Asp Met Met C XG TX T TY G YY AY A TG TO TG Arg His Leâ € ™ Val Ala G In Asp G Y AY Y T Z G TX G Y C C AM GA Y 0 l Phe Ser Val Tyr Met Asp Arg T-Y Q RS G TX T YY A TG G YY LG N Gly Gin Gl 1 USer 2-529570   The u Y T Z Phe T T Y Lys A M Gin C A M -Ser -Q R S Pro CX Gly G G X Tyr -   T Y Y A T H Gly G G'X Asp G A Y Asp GA Y Pro C CX Ile AT H Asn A Y Y Leu Y T Z Asn A Y Y Met Leu Thr Leu   G GX C A M -GA M Q RS A TG Y T Z A CX Y T Z   c Cy G Q C L T Gly Ala Asp Asp Ser Tyr Tyr   G G X G C X A T H G A Y C C X Q R S T A Y T A Y Thr Gly Ser Leu His Trp Pro Val   A C X GG X Q R YT Z C Y Y T G G T X C C X Val Thr Gln Gln Gln Tyr Trp Gln   GTX AC X G T X CA M CA M TA Y T G G CA M Phe Thr Asp Val Asp Ser Val Thr Ile   T Y AC X GT X G A Y Q R S G T X AC X AT H Ser Gly Vai Valley Val Ala Cys Glu   Q R S G G X G T X GT X G T X G C X T G Y G A M Gly Gly Cys Gln Leu Ala Island Asp   G G X G G X T G Y C A M G C X Y T H Y T Z G A Y Thr Gly Thr Ser Lys Leu Val Gly   AC X GG X A C X Q R Y Y M Y T Z G T X GG X Pro Ser Ser Asp Ile Leu Asn Ile   C C X Q R S Q R S G AT Y AT H YT Z AT Y AT H Gln Gln Ala Island Gly Ala Thr 'Gln   CA M G C X AT H GG X GC X A C X CA M Asn Gln Tire Gly Glu Phe Asp Island   AAY CA M T Y GG X Y M TT G Y A Y AT H Asp Asp Cys Asp Asn Leu Ser Tyr Met   GAY TG Y G A Y Y Y Y Z Q R S TA Y AT G Pro Thr Valley Val Phe Glu Ile Asn   C C X A C X G T X G T X T T Y G A M A T H A A Y Gly Lys Met Tyr Pro Leu Thr Pro   G G X A A M A T G T Y C C X Y T Z A C X C C X   961. Ser Ala Tire Thr Ser Gin Asp Gln   Q R S G C X T A Y A C X Q R S C A M G A Y C A M Gly Phe Cys Ser Ser Gly Phe Gln   G G X T T Y T G Y A C X Q R S G G X T T Y C A M   009 Ser Glu Asn His Ser Gln Lys Trp   QR S G A M AA Y C A Y Q R S C A M A A M TG G Leu Gly Island Asp Val Phe Arg Island   AT H Y T Z G G X G A Y G T X T T Y AT H LG N Glu Tyr Tire Ser Val Phe Asp Arg   G A M T A Y T A Y Q R S G T X Y T Y G A Y L G N Ala Asn Leu Val Leuly Leu Ala   G C X A Y Y Y Y T Z G T X G G X Y T Z G C X Ala Island Lily AA M G C X AT H in which the strand 5 'to 3', beginning with the amino terminus, and the amino acids for which each triplet codes, are represented, and in which the abbreviations have the   same meanings as in claim 3.   Calf chymosin gene according to claim 3, characterized in that it contains deoxyribonucleotide sequence Gly GGG Thr ACC Phe TTT Pro CCG Phe TTT Trp TGG Ser AG C Arg CG C Thr ACC Leu CTG Ser AGC Asp GACI comprises the following: Glu Val Ala Ser Val Pro GAG G T G G C C AG C G TG C C C Asn Tire Leu Asp Ser Gln A C TA C C T A G AG AG CAG Gly Lys Ile Tyr Leu Gly G G G A G AT G AT C TA C CT C GG G Pro Gln Glu Phe Thr Val C C C A G G T C C C T C Asp Thr Ser Gly Ser Asp GA C A C T GGC TC C T C T G A C Val Pro Ser Tire Cys Island G T I C C C T C A T T C T A C T G C Asn Ala Cys Lys Asn His A A T C C T G C A A A A A A C C A C Phe Asp Pro Arg Lys Ser   T T C G A C C CC G A G A A G T C G Phe Gln Asn Leu Gly Lys T T C C A G A C C T G G G C A A G Ser Isle His Tyr Gly Thr T C T A T C C A C T A C G G A C A Met Gln Gly Ile Leu Gly AT G C A G G C C G C G C G Thr Thr Val Val Asn Asn A C C G T C A C T G T C T C C A A C Val A 'p C T G G A C   1 Gln Gln Leu C T G Tyr T A C Thr A C C Leu C T G Phe T T C Lys A A G G C C G Ser T C C Pro C C C Gly G G C Tyr TAT Ile AT AT C C A G C A G AC A G T A G G C   Thr Val Gly 29- Ser Ser Thr Gln Glu Pro Gly Asp   C T G A G C A C C A G G G CC G G G G AC   Phe TTC ATC Leu CT C Val GTG His CA C Ser TCG Gln CAG Ala GCC Gly GGG Thr ACA Thr ACT Gly GGT Gly GGC Gly GGC Ser AGC Gln CAG Gln CAG Thr ACC Leu CTG Ala GCC Phe TT Leu CTG Val GTT Glu GAG Ile ATC Ser TCC Val GTG Val GTG Val GTG Cys TGT Thr AC C Ser 'AGC Ala GCC Tyr TAC Tyr TAT Gly GGG Ser TCA Asp GAC Val GTG Tyr TA C Ser. A CC Asp G A C Leu C T G Gln C A G Asp G A C Val G T T Gln C A G Ser T C C.   Asp GAC Ile ATT Gly GGT Ala GCN Met ATG Glu GAG Asn AA C Ala GCC Met ATG Met ATG Pro CCG His CA C Gln CAG Ser AGT Val GTG Ala GCC Lys AAG Ile ATC Gly GGA Glu GAG Glu GAA Ala GCC Tyr TAC Met ATG Gln CAA Asp GAC Leu CT C Ser TCC Trp TGG Tyr T A C -   Val GTC Ala GCC Ile AT C Leu CTG Leu CT C Ala GCC Phe TTT Phe TTC Tyr TAC Ser TCG Met ATG Asp GAC Arg AGG Thr ACG Tyr TAC Val GTG Trp TGG Thr ACC Cys TGT Leu CTG Val GTC Asn AAC Thr ACA Asp GAC Asn Leu Ser Tyr A C C T G AG C T A C   Asp G A C Pro C C C A T A Asn AA C Leu C T G Asn AA T Leu C T G Tyr T A C Pro CC C Gln C A G Ile AT C Glu GAG Asp G A C Gly G G G Ile AT C Gln C A G Ile A Tr C Met AT G Val -G T C   Gly G G G Ser C G Pro C C Arg AG G 601. Phe T T C Gly G C Gly GG G Thr A C A Val G T G Phe T T C Ser A G C Gly G G T Thr. A C G C C C C C C C G A C Asp Cys Asp Asp G A C TG C G A C Pro Thr Valley Val Phe Glu Ile Asn   CCC A C T G TG G T C T TC G A T C AAT Gly Lys Met Tyr Pro Leu Thr Pro   G G C A A AT G C C A C T G A C C CC C Ser Ala Tyr Ser Ser Gln Asp Gln   TCC G CC T A T AC C AG C C A A G CAG   -985 Gly Phe Cys Ser Ser Gly Phe Gln   G G C T T C T G T A CC A G T G G C -T T C C A G   Ser Glu Asn His Ser Gln Lys Trp AGC G A A AT C A T T C C T Ak AAAA TGG Ile Leu Gly Asp Val Phe Arg Island,   A TC C T G G G G G A TG T T T T C A T C C G A Glu Tyr Tire Ser Val Phe Asp Arg   GAG T AT TA C AG T C GT C TT T GA C AGG Ala Asr Asn Leu Val Gly Leu Ala   GCC A A C A C C C C T G GG C C G GCC Ala Island Lily A C C A C C A C   wherein strand 5 'to 3', starting with the amino terminus, and the amino acids for which each triplet codes, are represented, and in which the abbreviations have the   same meanings as in claim 3.   6 A calf prochymosin gene according to claim 4, characterized in that it comprises the following deoxyribonucleotide sequence: Ala Glu G C T G A G Isle Thr Arg Ile Pro Leu Tyr Lys   ATC AC C A G AT C C C T C T G T A C AA A Gly Lys Ser Leu Arg Lys Ala Leu   G C A A G T C T C T G AG G A A G G CT G Glu Lys His Gly Leu Leu Glu Asp   AAG G A G C A T GG G CT T C T G G A G GA C Phe Leu Gln Lily Gln Gln Tyr Gly   T C C T G C A G AA A C AG C AG T T T T G G C   Ser Ser Lys Is Tyr Ser Gly PI e A T C A G C A G C A A G T A C T C C G G C T l C 9 DI L   -71 as P aas 98 ú na-j 19 f D D D D 9 9 9 9 9 9 0.2 L T 7,   aqd D D v atil usv -4 w w w w ith 0-id dsv qqd d Aig AID IPA Garlic Ait) AID jqj, D v v old S Aq D D las jas UID STH   S D qj, ID lAj, UTE) niq 0.1 D t L -1 naq SÀU vvsv v I Kj, jas lqj, naq las D IV v usv D v D ni D 9 il v 1 la, q N Z) 9   VIV D v 1-7) and D D v D UID D 1 D TIPA D 1 IV garlic D v 1 -1, K J, E) 1 D   à v v v v v v v v v v v v v v v v v v v v v v 1 1 1 1 1 1 1 1 1 1 1 ID ID ID ID ID D 1 D jqj, AID UID S-CH gar v Usv UID D v 0-id CTSV D D S D LI 1 v D D D   jas D aqj, D v D n UID 1 v D vv a-EJ SÀ-1 he DD v 1 naq 2 AL 1 THE ri a 1 D CI qj ds V oid D vv usv D 1 DDDDD 1 DDD v DD 9 D 1 DD v DDDD e Jj -l A nie AID nai 01 CI TL e A the Tú ozs 6 zçz Arg His Leu Val Ala Gln Asp Leu   AGG C A C C TG G TG G C C CAA G AC CTG Phe Ser Val Tyr Met Asp Ars Asn   TTC T CG GT T TA C A T G GA C AG G AAT Gly Gln Glu Ser Met Leu Thr Leu   GG C AG G A G C AT G CT C A C G CT G Gly Ala Asp Asp Tyr Tyr Tyr   G G G G CA C T C A C C C C C T C C A T C Thr Gly Ser Leu His Trp Pro Val   A CA G G G T C C T T C G A CT T G G T C T C Val Thr Gln Gln Gln Tyr Trp Gln   GTG A C A G T G C A G C A G T A C T G G CAG Phe Thr Asp Val Asp Ser Val Thr Ile   TTC A C T G T G GA A G T G T C AC C AT C Ser Gly Valley Val Val Ala Cys Glu   A G C G G T G T G G T T G T G G C C TG T GAG Gly Gly Cys Gln Leu Ala Island Asp   G G T G G C T G T C A G G C C T C C T G G A C Thr Gly Thr Ser Lys Leu Val -Gly   ACG GG C AC C T C C A G C T G GT C GGG - Pro Ser Ser Asp Ile Leu Asn Ile   C C A G C A G C C A T C T C T C A T Gln Gln Ala Island Gly Ala Thr Gln   CAG C A G G C C C G G A G A C C A CA G Asn Gln Tire Gly Glu Phe Asp Island   A C C A G T A C G G T G A G T T T G A C T C Asp Asp Cys Asp Asn Leu Ser Tyr Met   G A C TG C GA C A A C T G A G C T A C AT G Pro Thr Valley Val Phe Glu Ile Asn   CCC A C T G TG GT T TT C G A G AT C AAT Gly Lys Met Tyr Pro Leu Thr Pro   GG C AAA AT C TA C C C C C C C A C C C C C Ser Ala Tyr Ser Ser Gln Asp Gln   T C C G C C T A T C C C C C C AA G A C C A G Gly Phe Cys Ser Ser Gly Phe Gln   GG C T C T G T A C C AG T G G C TT CC A G Ser Glu Asn His Ser Gln Lys Trp   AGC G AA A A T T C A T T C C AA A AT GG Leu Gly Island Asp Val Phe Arg Island   ATC C T G G G T GAT GTT T C AT C CGA Glu Tyr Tire Ser Val Phe Asp Arg   GAG T AT TA C AG T C G C T T GA GA AG Ala Asn Leu Val Leuly Leu Ala   GCC AA C A A C C C C T G G G G C T G C C C Lily Ala -Ile AAA G C C AT C   wherein strand 5 'to 3', starting with the amino terminus, and the amino acids for which each triplet codes, are represented, and in which the abbreviations have the   same meanings as in claim 3.   A calf chymosin gene according to claim 1, characterized in that it comprises the following deoxyribunucleotide sequence: Cys Leu Val Val Leu Leu Leu Ala Val   TGT CT C G T G G T G C T A CT T G CT GT C Phe Ala Leu Ser Gln Gly Ala Glu   TTC CT CT C T C C CAG GG C G C T GAG Isle Thr Arg Ile Pro Leu Tyr Lys   ATC AC C AGG AT C C CT C T G T A C AAA Gly Lys Ser Leu Arg Lys Ala Leu   G C A A G T CT G G G A G G C G CTG Glu Lys His Gly Leu Leu Glu Asp   AAG G A G C A T GG G CT A CT G G A G GA C Phe Leu Gln Lily Gln Gln Tyr Gly   T C C T G C A G A A A C A G C A G T A T G G C Ser Ser Ser Lys Tyr Ser Gly Phe   A T C A G C A G C A A G T A C C C G G C T T C Gly Glu Val Ala Val Val Pro Leu   GGG GAC GTG G C C AG C G G C C C T G Thr Asn Tire Leu Asp Asp Gln Tyr   AC C AA C TA C CT A G AT AG T C AG TA C Phe Gly Lys Island Tyr Leu Gly Thr   TT GG G AA G AT C TA C C T C G G G AC C Pro Pro In Glu Phe Thr Val Leu   CGC CCC C A G G A G T T C C C G TG CTG   Asp GA.C Val GTA Asn AA T Phe TTC Phe TTC Ser TCT Met AT G Thr AC C Asp GAC Ser Phe TTC TTC ATC Leu CT C Val G TG His CA C Ser TCG Gln CAG Thr ACT ACT Pro CCC Ala GCC Asp GAC Gln CA G Ile AT C Gln CA G Val GTC Ile AT C Thr t Ae e Thr AC C Leu CTG Ala GCC Phe TTT L.eu CTG Val GTT Glu GAG Gly GGC Ser TCA Cys TGC Pro C CG Asn AA C His CA C Gly GGC Thr ACT ACT Gln CAG Gln eC Tyr TAT Gly GGG Ser TCA Asp GAC Val GTG Tyr TAC Ser AGC Ser TC C Ile ATC Lys AAA Arg AGA Leu CTG Tyr TA C Ile ATC Val GTC Gln CAG Glu Gi u -G A- Ala G CN Met ATG Glu GAG Asn AAC Ala GCC Met AT G Met ATG Ser TCT Tyr TAC Asn AAC Lys AAG Gly GGC Gly GGG Leu CTG Ser TC C Thr ACA Pro Glu GAA Ala GCC Tyr TAC Met ATG Gln CAA Asp GAC Leu CT C Asp GAC Cys TGC His CA C Ser TCG Lys AA G Thr ACA Gly GGC Asn AAC Val GTA Gly GG Phe TTC Tyr T'A C Ser TCG Me t ATG Asp GAC Arg AGG Thr ACG Phe TT C Lys A G G C C G Ser TC C Pro C C-C Gly GG C Tyr T.A T T T Gly G G C Asp G C Asp G A C Pro C C C Ile AT A Asn AA C Leu C T G Asn A AT Leu C T G Ala Ile Asp Pro Ser Tyr Tyr GC C AT C GA C C C TC TC C AT C TA C   Phe TTT Trp TG G Ser AG C Arg CGC Thr ACC Leu CTG Ser AG C Asp GAC Val G TG Leu "-G Val G T C Gly G G Ser T C G Pro CCC Ars AG G Phe T T C Gly G G C Gly GGG -2529570 Thr Gly Ser Leu His Trp A CA G G G T C C C T G C A C T G G Val Thr G T G A C A Phe Thr T T C ACT Ser G ly AG C G G T Gly Gly G G T G G C Thr Gly AC G G G C Pro Ser C C C AG C Gln Gln C A G C A G Asn Gln A C C A G Asp Cys G A C T G C Pro Thr C C C A C T Gly Lys G G C A A A Ser Al - T C C G C C Gly Phe G G C T T C Ser Glu A G C G A A. Ile Leu AT C C T G Glu Tyr GAG TAT   Val GTG Val GTG Val GTG Cys TGT Thr ACC Ser AG C Ala GCC Tyr TAC Asp GAC Val GTG Met ATG Tyr TAT Cys TGT Asn AAT Gly GGG Tyr TAC Gln CAG Asp GAC Val GTT Gln CAG Ser TCC Asp GAC Ile ATT Gly GGT Asn AAC Val GTC Tyr TAC Thr AC C Thr AC C His CAT Asp GAT Ser AGC Ala Asn Asn Leu GCCC A C A C C T C G   Gln CAG Ser AG T Val GTG Ala GCC Lys AAG Ile AT C Gly GGA Glu GAG Leu CTG Phe TTC Pro CCA Ser AGC Ser AGT Ser TCC Val GTT Val GTC Tyr TAC Val GTC Ala GCC Ile ATC Leu CTG Leu CT C Ala GCC Phe TTT Ser AGC Glu GAG Leu CTG Gln CAA Gly GGC Gln CAA Phe TTC Phe TT T Val Pro CCC GTG   Trp T G G Thr AC C Cys- T G T Leu C T G Val G T C As-n A A C-   Thr ACA Asp GAC Tyr TAC ATC Thrac AC C Asp GAC Phe TTC ATC lily ATC Asp GAC Gin CAG ATC Gluc GAG Asp GAC Gly GGG ATC Gln CAG ATC ATG Asn AAT Pro CCC Gln CAG Gln CAG Trp TGG Arg CGA Arg AGG Val Gly Leu; i T G G G C T G G C Ala Island Lily   AAA G C C AT C T CA T CA C AT G C TGA   C CA A G A C C T CA C TG T CC C C A C A C   A C C T C C A CA C A C A CA T CA C CAT   G TA C AT G A G CAC ATG TGC AT C A C A C   A C G A T G AG T G TT T CC A G A C A G A T G   ATT C T C A A T AA C G T T T C T T T G AAA AAA AAA AA AA   wherein the strand 5 'to 3', starting with there amine termination, and the amino acids for which each triplet codes, are represented, and in which the abbreviations have the   same meanings as in claim 3.   Plasmid having the property of replication in a prokaryotic organism, characterized in that it comprises a deoxynucleotide sequence coding for chymosin of calf.   Plasmid having the property of replication in a prokaryotic organism, characterized in that it comprises the calf chymosin gene according to any of the Claims 1 to 7.   Plasmid according to Claim 9, characterized   in that saidprocaryotic organism is of the genus Escherichia.   11 Microorganism transformed by the plasmid of claim 9.   Microorganism according to claim 11, of the Escherichia kind.   Microorganism according to claim 12, of the species coli.   Process for the production of a substantially pure calf prochymosin, characterized in that it consists in cultivating, on an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals and growth factors, under conditions adapted to produce calf prochymosin, a prokaryotic organism transformed by a plasmid capable of replication in said organism and having a deoxynucleotide sequence coding for calf prochymosin; and to recover the   prochymosin of veal thus produced.   Method according to Claim 14, characterized   in that the prokaryotic organism is Escherichia coli.   Method according to claim 15, characterized in that said prokaryotic organism is substantially similar at N B-15061 of NRRL.   17 Biologically pure culture of a strain of   E coli p Gx 1225, N B-15061 of NRRL.