RECOMBINANT PLASMID AND MICROORGANISM CONTAINING SAME

Abstract: Messenger RNA of prorennin is extracted from the fourth stomach of calf and the double-stranded prorennin cDNA is prepared thereform. The prorennin cDNA is cloned into a host microorganism, E. coli C600rm, to give clones containing the cDNA insert with variable size as confirmed by the base sequence determination in addition to the colony hybridization and hybrid-arrested translation technique. In vitro recombination of two plasmids obtainable from these clones, pTACR102B6 and pTACR103C2, affords plasmid pCR1001 containing the whole coding sequence for proren­ nin. On additon of the lac operon of E. coli to pCR1001, plasmid pCR2001 is prepared, which plasmid contains the prorennin cDNA sequence and the promotor requisite for the expression of the prorennin gene in a host cell. Transforma­ tion of E. coli C600rm by inserting pCR2001 produces a new E. coli strain, E coli CR1 fully designated as E. coli CR1(r m, thr⁻, leu⁻, thi⁻, pCR2001). This genetically engineered new strain of E. coli, when propagatd in aqueous nutrient media, produces prorennin. Thus production by bacteria of proren­ nin which is a proenzyme and a precursor of the milk coagulating enzyme, calf rennin, has now been accom­ plished.

Description: RECOMBINANT PLASMID AND MICROORGANISM CONTAINING SfLnlE This invention relates to a recombinant plasmid and a host microorganism containing same, which microorganism is capable of producing calf prorennin. More specifically, this invention relates to a recombinant plasmid comprising cDNA(cor:iplementary DNA) of calf prorennin. Calf prorennin is a proenzyme protein secreted by the fourth stomach of the calf within about a few weeks after birth, and it is the precursor of the milkcoagulating enzyme, rennin(chymosin), indispensable in a process for making cheese. Rennin is excreted from the mucosa of the fourth stomach of calf and is an acidic protease having 323 amino acid residues. Prorennin, the precursor of rennin, is an enzyme protein having 365 amino acid residues and a molecular weight of about 40,000 and it is converted to the milkcoagulating enzyme rennin autocatalytically under acidic conditions. In recent years, the production of rennin has not been albe to meet demands, and a substitute microbial rennet is being used. Accordingly, there is great industrial significance in the supply of a large amount of rennin or prorennin. Recombinant DNA technology refers to the technique of introducing into the vector-plasmid of a suitable host microorganism, cDNA having genetic information by which an useful substance is produced; forming a recombinant plasmid; and utilizing said host organism containing the recombinant plasmid to produce said useful substance. Such recombinant DNA technology has progressed rapidly in recent years, and for instance, a technique of producing useful substances such as growth hormone, interferon, etc. by Escherichia coli as the host microorganism is under development. Beppu, one of the present inventors, and others have been investigating the cloning of the structural gene of prorennin in all sequences. The works have been disclosed in the following papers; "Abstracts" of the Annual Meeting of the Japanese Agricultural Chemistry Society," p408 in 1980, Proceedings of the IVth International Fermentation Symposium,, pl79 F-21-5(L) in 1980, "Abstracts" of the Annual Meeting of the Japanese Agricultural Chemistry Society, p259 and p569 in 1981, and Agric. Biol. Chef. 44 (6), p. 1373-1381 (1980). However, the present inventors have not successfully completed the cloning of clones which have essentially all the sequence of the structural gene of prorennin and are expected to be capable of producing calf prorennin, the presence of which clones can be confirmed by the hybrid-arrested translation assay method. In the clonings in the above-reported literatures, strains displaying positive hybridization have been obtained; nevertheless, they do not contain the entire cDNA of prorennin but the partial segment of the cDNA having the partial sequence of the stuctural gene of prorennin with a length of about 0.2Md. As a result of further studies that will be explained in detail in Examples of this specification, the inventors have now succeeded in preparing clones which contain a recombinant plasmid having essentially all the sequence of the prorennin structural gene as confirmed in the hybrid-arrested translation assay and are capable of producing calf prorennin. The inventors, using the method that will be detailed later, have succeeded in constructing a recombinant plasmid comprising calf prorennin cDNA and in obtaining host microorganisms having this recombinant plasmid. Novel microorganisms having this novel recombinant plasmid are those belonging to the P2 level (deposition of which is not accepted by Fermentation Research Institute, Agency of Industrial Science and Technology Japan) and one species, for instance, Escherichia coli C 600 rkmk (pTACR1), which is stored at Fermentation Laboratory, Agricultural Chemmistry Department, Faculty of Agriculture, the University of Tokyo. The purpose of this invention is to provide a recombinant plasmid comprising calf prorennin cDNA and furthermore, a host microorganism having said recombinant plasmid. Another purpose of this invention is to provide a process for producing calf prorennin which comprises cultivating in a nutrient medium, a genetically engineered microorganism and isolating calf prorennin from the culture broth, said genetically engineered microorganism containing a recombinant plasmid composed of calf prorennin cDNA. The above purposes of this invention as well as advantages thereof will become clearer in the following description. According to this invention, prorennin mRNA (messenger RNA) is extracted from the fourth stomach of the calf within a few weeks after birth and a complete mentary single-stranded DNA is synthesized using the reverse transcriptase method with said mRNA as template, which template is then removed by alkaline treatment. From the single-stranded DNA obtained above, a doublestranded cDNA is synthesized using, for instance, DNA polymerase I or reverse transcriptase. By the method utilizing nuclease S1, only the hair-pin structure is removed selectively from the double-stranded cDNA to form the double-stranded cDNA. Then, to both 3' ends thereof is attached poly dG(deoxyguanine) homopolymer according to the TdT(terminal deoxynucleotidyl transferase) method. On the other hand, a suitable vector plasmid such as E. coli vector pBR322 is treated with restriction enzyme, Sal I to furnish linear pBR322 DNA and then both 3' ends of a single-stranded portion of the linear pBR322 are repaired by using the DNA polymerase I method. To the repaired 3' ends is attached poly(dC)homopolymer according to the TdT method. The linear pBR322 DNA with the attachement of poly(dC)homopolymer at the 3'ends is mixed with the double-stranded cDNA having the attachement of poly(dG)honopolymer at the 3' ends, and the mixture is annealed to form a recombinant plasmid comprising calf prorennin cDNA. Then, E. coli is treated with calcium salts, preferably at a cold temperature and mixed with the recombinant plasmid to form transformant strains of E. coli. Clones having some inserted DNA at the Sal I site are selected, according to the method utilizing drug-resistance of vector plasmids. In order to choose clones having prorennin cDNA among the selected ones above, colony hybridization is performed using as probe a radioactively-labelled proprennin-specific mRNA such as 1251-mRNA or 32P-mRNA. Candidate strains of host microorganism possibly comprising calf prorennin cDNA are screened by the hybrid-arrested translation method for the presence of the inserted structureal gene of prorennin. As will be shwon in the Examples later, the present invention has provided clones having substantially all the sequence of the prorennin structural gene as confirmed by the hybrid-arrested translation method. Furthermore, according to the method of Maxam and Gilbert, the presence of base sequence coresponding to the amino acid sequence of prorennin N terminus has been confirmed. Although in the preceding descrption the inventors have disclosed one embodiment of obtaining recombinant plasmid and host microorganism containing same, several variations can be adopted with respect to the embodiment, such as in choosing host microorganism or vector plasmid and in synthesizing recombinant plasmid. As other examples of the host microorganism, yeast such as Saccharomvces cerevisiae, gram-positive bacteria (eg. Bacillus subtilis), and gram-negative bacteria (eg. Pseudomonas) can be exemplified. For instance, the method for synthesizing a recombinant plasmid using a restriction enzyme, endonuclease-ligase, is also applicable in this invention. Other examples of the vector plasmid are as follows: when E. coli is the host microorganism, known vector plasmis such as pMB9 are used; when the host is yeast, various known vector plasmids derived from 2;DNA or yeast chromosone are used; when the host is B. subtilis, known vector plasmids such as pUB101 are used; and when the host is Pseudomonas bacterium, various plasmids having drugresistance are used. An embodiment of this invention will be illustrated further in the following. I. Isolation of Prorennin mRNA Prorennin mRNA is extracted from the mucosa of the fourth stomach of a calf within about a few weeks after birth. Extraction can be carried out acceding to a modiciation of Uchiyama's mehod (H. Uchiyama, T. Uozumi, T. Beppu, and K. Arima, Agr. Biol. Chew. 44, p. 1373-1381, 1980). For instance, the mucosa of the fourth stomach of calf are collected and powdered at low temperature, from which all RNA is extracted by the phenol method, preferably in the presence of bentonite. An aqueous extract containing polymer RiNA is treated with a precipitating agent such as lithium chloride and the precipitate containing the polymer RNA is collected. From an aqueous solution containing the polymer RNA precipitates thus prepared, poly(A) tailed RNA fractions containing prorennin mRNA can be separated and collected by affinity-chromotography using an absorbent such as poly(U)-Sepharose or oligo(dT)Sepharose. Namely, the poly(A)mREJA fractions containing prorennin mRNA are selectively adsorbed on the adsorbent, and ribosomal RNA and other impurities are eluted and removed through the adsorbent column. Elutdion with an aqueous formamide solution gives an aqueous formamide solution containing prorennin mRNA. Preferably, the above-stated operations are repeated several times. In accordance with a known procedure, the fractions containing prorennin mXlA are subjected to the sucrose density gradient centrifugation and by such molecular weight fractionation, fractions (prorennin mRNA fractions) having a molecular weight centered at a sedimentation constant of 15s can be collected. The mRNA fractions are tested by in vitro protein synthesis for their coding activity. These fractionating operations can be repeated a number of times if desired. II. Preparation of Prorennin cDNA Using the reverse transcriptase method with the above prorennin mZJA as template, the complementary single-stranded DNA of said mXlA is synthesized and the single-stranded cDNA of prorennin can then be obtained after removal of the template mRNA by alkaline treatment. For instance, said prorennin mZlA is reacted with the reverse transcriptase in the presence of four kinds of deoxynucleotide triphosphates (dXTP's) and also oligo(dT) as a primer, and the complementary singlestranded DNA complexed with said prorennin mRNA can be synthesized. The resulting material is heated at around 700C in the presence of an alkali to remove the template mRNA by destruction and thus the single-stranded prorennin cDNA is produced. The single-stranded prorennin cDNA is then reacted with DNA polymerase I or a reverse transcriptase in the presence of dXTP's to form a complementary double-stranded cDNA. Since the double-stranded cDIJA has a hair-pin structure, the cDNA is reacted with nuclease S1 that is capable of selectively cutting off the hairpin structure and after removal thereof, the complete prorennin-specific double-stranded cDNA can be synthesized. III. Libation of Prorennin cDNA and Vector Plasmid Cor:plernentary deoxynucleotidyl homopolymer is attached to botn 3' ends of the above synthesized complete prorennin-specific double-stranded cDNA. A suitable hcst vector plasmid is cut at desired sites and to the resulting linear DNA is attached the homopolymer at both 3' ends. For instance, poly(dG)homopolymer can be attached to both 3'ends of said prorennin double-stranded cDNA by the reaction with TdT in the presence of dGTP(deoxyguanosine triphosphate). On the other hand, a suitable vector plasmid such as vector plasmid pBR322 of E. coli is reacted with restriction enzyme Sal I endonuclease to form linear pBR322 DNA. Both 3'ends of the single- stranded portion of the linear plasmid DNA are repaired by the reaction with DNA polymerase I in the presence of four kinds of deoxynucleotide triphosphates (dXTP's) and to the repaired ends can be attached poly (dC) homopolymer by the reaction with TdT in the presence of deoxycytosine triphosphate (dCTP). According to this invention, a recombinant plasmid containing calf prorennin cDNA can be synthesized in the following manner. The prorennin-specific double-stranded cDNA having poly(dG)homopolymer attached at 3'ends such as the one prepared above, is mixed with the linear pBR322 DNA having poly(dC)homopolymer attached at the repaired 3'ends, and upon annealing the mixture both DNA are linked at the poly(dC) tail of the latter DNA and the complementary poly(dG) tail of the former. Thus the recombinant plasmid containing essentially the entire length of the prorennin structural gene can be synthesized. IV. Transformation of Host Microorganism and Detecticn of Clones Containing Recombinant Plasmid Comprising Calf Prorennin cDNA A host microorganism such as E. coli C 600 r m is treated with calcium salts and then mixed with the recombinant plasmid comprising calf prorennin cDNA to incorporate said recombinant plasmid into E. coli, thus transformants being prepared. Clones with the recombinant plasmid comprising prorennin cDNA are selected from the transformants. For instance, when prorennin cDNA is inserted at the Sal I site of pBR322, the transformants with the recombinant plasmid comprising calf prorennin cDNA are expected to be ampicillin-resistant and tetracyclinesensitive. In a preliminary manner, the desired transformants can be distinguished from those that are ampicilin-resistant and tetracycline-resistant due to the differnece in resistance against ampicillin and tetracycline. In order to separate the real transformants having the recombinant plasmid from those that are expected to have same, colony hybridization and hybridarrested translation are carried out. The colony hybridization can be performed according to a known method where the prorennin-specific mRNA is labelled with radioactive 1251 or 32p and 125I RN or 32P-mRNA is used as probe. By this colony hybridization, clones displaying positive hybridization are identified and they are further tested by the known hybrid-arrested translation for the presence of the inserted structural gene of prorennin. The following examples will exemplify the pratice of the invention. They are presented for illustrative purposes only, and should not be construed as limiting the invention in any way. Example (1) Extraction and Purification of Prorennin Messenger RNA (mRNA) Mucous membrane of two calf abomasums (two weeks after birth) (ca. 140g) was peeled off; wahsed with a cooled Tris-HcQ buffer solution (0.lM, pH9.0) containing 0.025 M EDTA, 0.1M NaCl, and 1% SDS; dehydrated in acetone-dry ice; frozen; and powdered with a homogenizer. The powders were suspended in the buffer solution (1L) containing bentonite and to this was added a mixed solvent (1Q) comprising phenol, chloroform, and isoamyl alcohol (50 : 50 : 1) and after stirring, it was centrifuged and the upper aqueous phase was separated. After repeating same operations six times, lithium chloride was added to the final aqueous phase to make the final concentration 2M. Upon standing overnight at 40C, precipitates were collectd by centrifugation and they were dissolved in 60ml of 0.1 x SSC (150mM of NaCl and l5mM sodium citrate). The same operations were repeated once again and to the resulting solution were added two volumes of ethanol to form precipitation of polymer RNA, which was repeated three times. The final precipitate was dissolved in water (6 mQ) and to this was added 4 volumes of a Tris-HcL buffer solution (pH 7.5) containing 0.01M EDTA1 0.7M NaCl, and 25% formamide. The solution was passed through poly (U) Sepharose 4B column (volume, 30ml) and the column was washed with the above buffer solution (30ml). After washing with 30ml of the buffer solution, the adsorbed mRNA was eluted with an aqueous solution (pH 7.5) containing 0.01M EDTA, 0.2% SDS, 90% formamide, and 0.01M potassium phosphate. The eluate was dialysed against distilled water and on addition of ethanol, zJA containing m;A was precipitated. The precipitate was, after dissolving, centrifuged for 13 hours at 50,000 x g in 5 to 20% sucrose density gradients. Fractions containing prcrennin mRNA (120Pg of RNA with a molecular weight centered at 15s) were collected. These fractions were tested in vitro in protein synthesis system using rabbit reticulocyte lysate- and it was confirmed that the fractions have such coding activity that can produce prorennin and the amount thereof exceeds 70% of the total translational products. (2) Preparation of Prorennin cDNA In order to synthesize single-stranded cDNA by using the prorennin mPlA as template, a solution (400 l) containing the following ingredients was incubated at 42 C for lh; these are mRNA (30 l), AMV reverse transcriptase (140 units), oligo(d.)12 - 13 (1.6 P), four kinds of deoxynucleotide triphosphates (each 0.5mM), Actinomycin D(40 g), NaCl(60mM), MgC12(6mM), DTT(2mM), and Tris-Hcl (50mM, pH8.3) The reaction mixture was extracted with phenol, precipitated with ethanol, dissolved in 0.3N NaOH, and heated at 680C for 15 min to destroy the template mRNA. Subsequent gel filtration through Sephadox G-50 gave 240 ng of the single-stranded cDNA. In order to synthesize double-stranded cDNA by using the above single-stranded cDNA, a solution (240 u1) containing the following ingredients was incubated at 250C for 4h; these are the single-stranded cDNA (200 ng), DNA polymerase fragment A (18 units), four kinds of deoxynucleotide triphosphates (each lmM), MgC12(4mM), S-mercaptoethanol (0.SmM), and Tris-HCl (30mM, pH7.5). The reaction product was extracted with phenol and precipitated with ethanol. The precipitates were treated with 80 units of nuclease Se at 400C for 90 min in a solution (200 1, pH 4.6) containing sodium acetate (30mM), NaCl (S0mM), and ZnSO4 (lmM) and the hair-pin structure were removed by decomposition. The resultant product was centrifuged in from 5 to 30% sucrose density gradients at 95,000 xg for 12h to produce 100 ng of the double-stranded cDNA having a mean molecular weight of 0.7 Md. (3) Annealing of Prorennin cDNA and Vector Plasmid The above prorennin cDNA(0.05 p mole) was incubated at 37eC for 6 min in a solution A (120 ul) containing 80 units of terminal deoxynucleotidyl transferase, dGTP(0.06 mole), MgC12(5mM), and HEPES NaOH (lOmM, pH7.1); and (dG) homopolymer was thus attached to 3' ends of the cDeJA. On the other hand, vector plasmid pBR322 was cut by Sal I, endonuclease to form linear DNA and the linear DNA was incubated with DNA polymerase(fragment A) in the presence of four kinds of deoxynucleotide triphosphates to repair the single-stranded ends. The DNA thus obtained (0.5 p mole) was incubated at 250C for 10 min in a solution B (120 1) containing 24 units of terminal deoxynucleotidyl transferase, dCTP (0.04 p mole), CoC12(lmM), potassium cacodylate(l00mM), and Tris-HCl(30mM, pH7.6); and (dC)homopolymer was thus attached to 3' end of the DNA. Reaction mixture A (120 1) and reaction mixture B (24 1) were mixed together at 0 C after stopping incubations in both mixtures A and B by the addition of EDTA. The product was extracted with phenol and precipitated with ethanol. The precipitate was dissolved in a l00mM Tris-HCl solution (pH 7.6) containing EDTA(lmM) and NaCl (l00mM) at a concentration of 0.3 ug per ml and it was heated at 680C for 2h and gradually cooled to room temperature at a cooling rate of 5 C per 30 min. Thus the prorennin cDNA having (dG) homopolymer attached and pBR322 DNA having (dC) homopolymer attached were linked by annealing. (4) Transformation of E. coli with Annealed DNA and selection of Clones Containing Prorennin cDNA Transformation of E. coli by the use of the annealed DNA obtainable in process (3) was performed according to a conventional method involving the use of calcium treatment. E. coli C600r m transformed accordingly and 804 transformants were selected on L broth agar plates containing 30 p9/ml ampicillin. Then, among 804 colonies, transformants that were ampicillin-resistant and tetracycline-sensitive on the same medium containing 10 vg/ml of tetracycline were selected. The total number of such colonies was 622. The 622 transformants were implanted on Millipore filters in a lattice fashion that were attached onto the L broth agar medium containing 30 ug/ml of ampicillin. Colonies grown on the filters were washed, treated with protease, and heated in vacuo at 800C for 2h after teatment with 95% ehanol and chloroform, thus fixing DNA of each colony onto the filters. Three filters were prepared simultaneously and each filter was subjected to colony hybridization in the following manner; filter (1) was hybridized with the prorennin mRNA labelled with 1251 as probe; filter (2) was, after treatment with excessive ribosomal rRNA, hybridized with the same 125I-mRNA; and filter (3) was hybridized with 125I-rRNA. By comparing all three filters, clones containing rDNA were first removed and thirty clones presumably containing prorennin cDflA were obtained. Recombinant plasmids (DNA) were obtained from these thirty clones by CsCl-ethidium bromide equilibrium centrifugation. Each plasmid was hybridized with the prorennin mRNA obtained from the previous process (1) and tested for in vitro protein synthesis in a reticulocyte lysate system to see if it inhibits the prorennin synthesis. By this hybrid-arrested translation method, ten clones were determined to contain the prorennin cDNA. (5) Characteristics of the Cloned Prorennin cDNA pTACR1 plasmid DNA was extracted from one of the clones (102-3 strain), treated with Sat a I endo- nuclease, and subjected to agarose gel electrophoresis. Thus inserted DNA fragment corresponding to approximately the entire length of prorennin cDNA was detected to be present. The base sequene of this inserted DNA fragment was determined according to the method of Maxam and Gilbert. As will be evident in the following, the base sequence corresponding to the amino acid sequence prorennin N terminus has been proved to be present in the inserted DNA fragment. 5'-GCT GAG ATC ACT AGG ATC CCT CTG Ala Glu Ile Thr Arg Ile Pro Leu TAC AAA GGC-3 Tyr Lys Gly The plasmid DNAs obtained from the clones definitely containing the prorennin cDNA was analyzed according to the Maxam-Gilbert method for its base sequence. As a result of the base sequence determination, it was found that out of 1095 bases corresponding to the entire sequence of the prorennin cDNA, plasmid pTACR 102B6 comprises from the 1st base corresponding to the prorennin N terminus up to the 890th base and plas mid pTACR 103C2 comprises from the 771st base up to the 1095th base corresponding to the prorennin C terminus. Plasmid pCR 1001 containing the entire length of prorennin cDNA was constructed in the following manner. pTACR 102B6 and pTACR 103C2 were treated with Sal 1 to obtain DNA fragments. Each DNA fragment was digested with exonuclease to cut off about 150 bases from both 3'ends. The fragment derived from pTACR 102B6 was cut with Kpn I and the fragment derived from pTACR 103C2 was cut with Barn HI, respectively. Both fragments were annealed thereafter to produce fragment (a). Independently, small fragment (b) was prepared by the treatment of pTACR 102B6 with EcQ RI plus Kpn I. In a similar manner, large fragment (c) was prepared by the treatment of pTACR 103C2 with Eco RI plus Bam HI. All three fragments, (a), (b) and (c) were mixed, annealed and linked by T4 DNA ligase. pCRlO0l thus constructed contains the complete cDNA corresponding to all the amino acid sequence of prorennin. The base sequence including the stopping codon thereof is illustrated below. 1 20 40 GCT GAC ATC ACC AGC ATC CCT CTG TAC AAA GGC AAG TCT CTG AGG AAG GXG CTG AAG Ala Glu Ile Thr Arg Ile Pro Leu Tyr Lys Gly Lys Ser Leu Arg Lys Ala Leu Lys 1 10 60 80 100 GAC CAT GGG CTT CTG GAG GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGG AGC AAG Glu His Gly Leu Leu Glu Asp Phe Leu Gln Lys Gln Gln Tyr Gly Ile Ser Ser Lys 20 30 120 140 160 TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GAT AGT CAG Tyr Ser Gly Phe Gly Glu Val Ala Ser Val Pro Leu Thr Asn Tyr Leu Asp Ser Gln 40 50 180 200 220 TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG CCC CAG GAG TTC ACC GTG CTG TTT GAC Tyr Phe Gly Lys Ilu Tyr Leu Gly THr Pro Pro Gln Glu Phe Thr Val Leu Phe Asp 60 70 240 260 280 ACT GGC TCC TCT GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT GCC TGC AAA Thr Gly Ser Ser Asp Phs Trp Val Pro Ser Ile Tyr Cys Lys Ser Asn Ala Cys Lys 80 90 300 320 340 AAC CAG CAG CGC TTC GAC CCG AGA AAG TCG TCC ACC TTC CAG AAC CTG GGC AAC CCC Asn His Gln Arg Phe ASP Pro Arg Lys Ser Ser Thr Phe Gln Asn Leu Gly Lys Pro 100 110 360 380 CTG TCT ATC CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TAT GAC ACC GTC Leu Ser Ile His Tyr Gly Thr Gly Ser Met Gln Gly Ile Leu Gly Thr Asp Thr Val 120 130 400 420 440 ACT GTC TCC AAC ATT GTG GAC ATC CAG CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC Thr Val Ser Asn Ile Val Asp Ile Gln Gln Thr Val Gly Leu Ser Thr Gln Glu Pro 140 150 460 480 500 GGG GAC GTC TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG GCC TAC CCC TCG Gly Asp Val Phe Thr Tyr Ala Glu Phe Asp Gly Ile Leu Gly Met Ala Tyr Pro Ser 160 170 520 540 560 CTC GCC TCA GAG TAC TCG ATA CCC GTG TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG Leu Ala ser Glu Tyr Ser Ile Pro Val Phe Asp Asn Met Met Asn Arg His Ley Val 180 190 580 600 620 GCG CAA GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GAC AGC ATG CTC ACG Ala Gln Asp Leu Phe Ser Val Tyr Met Asp Arg Asn Gly Gln Glu Ser Met Leu Thr 200 640 660 680 CRG GGG GCC ATC GAC CCG TCC TAC TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA Leu Gly Ala Ile Asp Pro Ser Tyr Tyr Thr Gly Ser Leu Bis Trp Val Pro Val Thr 210 220 700 720 740 GTG CAG CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC AGC GGT GTG GTT GTG Val Gln Gln Tyr Trp Gln Phe Thr Val Asp Ser Val Thr Ile Ser Gly Val Val Val 230 240 760 780 GGC TGT GAG GGT GGG TGT CAG GCC ATC CTG GAC ACC GGC ACC TCC AAG CTG GTG GGG Ala Cys Glu Gly Gly Cys Gln Ala Ile Leu Asp Thr Gly Thr Ser Lys Leu Val Gly 250 260 800 820 840 CCC AGC AGC GAC ATC CTC AAC ATC CAG GAG GCC ATT GGA GCC ACA CAG AAC CAG TAC Pro Ser Ser Asp Ile Leu Asn Ile Gln Gln Als Ile Gly Ala Thr Gln Asn Gln Tyr 270 280 860 880 900 GAT GAG TTT GAC ATC GAC TGC GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG Asp Glu Phe Asp Ile Asp Cys Asp Asn Leu Ser Tyr Met Pro Thr Val Val Phe Glu 290 300 920 940 960 ATC AAT GGC AAA ATG TAG CCA CTG ACC CCC TCC GCC TAT ACC AGC CAA GAC CAG GGC Ile Asn Gly Lys Met Tyr Pro Leu Thr Pro Ser Ala Tyr Thr Ser Gln Asp Gln Gly 310 320 980 1000 1020 TTC TGT ACC AGT GGC TTC CAG AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT Phe Cas Thr Ser Gly Phe Gln Ser Glu Asn His Ser Gln Lys Trp Ile Leu Gly Asp 330 340 1040 1060 1080 GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AAC AAC CTG CTG GGG CTG Val Phe Ile Arg Glu Tyr Tyr Ser Val Phe asp Arg Ala Asn Asn Leu Val Gly Leu 350 360 1095 GCC AAA ACC ATC TGA Ala Lys Thr Ile * 365 Linking of pTACR102B6 and pTACR103C2 can also be accomplished in the manner presented below. Namely, the linking employs plasmid pTACR5 as a joint portion which plasmid contains from the base site before Kpn I site (from No. 249 to 254) up to the site after Bam HI (from No. 1013 to 1018). pTACR5 was derivable from one of the ten prorennin cDNA containing clones obtained in process (4). DNA fragment (d) to be inserted was prepared by the treatment of pTACR 102B 6 with Hind III plus KDn I. Fragment (e) containing 760 bases (plus chain side) was prepared by the treatment of pTACR 5 with Kcn I plus Bam HI through its partial digestion. Fragment (f) was prepared by the treatment of pTACR103C2 with Bam HI plus Hind III. The three fragments possess termini complementary to each other and these were annealed to form the linear DNA comprising (d)-(e)-(f). After annealing, the cyclic DNA pCR1001 was constrjcted by treatment with T4 DNA ligase. The complete prorennin cDNA contained in plasmid pCR1001 was linked to te lac promoter region of E. coli lactose operon, with the frame fixed and thus pCR2001 was constructed. pCR2001 was inserted neo E. coli C600r :n by transformation. The plasmidcontaining microorganism is designated as E. coli CR1(rm, three leu , thi ; pCR2001) and has been deposited with the ATCC as ATCC No. 39170 (deposited on August A, 1982). The construction of pCR2001 is illustrated below in detail. (1) Plasmid pBH20 [K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F. Bolivar, H. W. Boyer; Science, 198, 1056 (1977)] contains the lac promoter of E. coli lactose operon, the ribosome-binding site thereof and a part of N terminus of a-galactosides. Plasmid pBH20 was treated with Eco RI to form a linear DNA, which was then subjected to DMA polymerase I. The terminal single-stranded DNA portion thus becomes doule-stranded, Decanucleotide containing Bam HI site was linked to the DNA at its both ends by the action of T4-DNA ligase and the DNA was further treated with Bam HI plus Sal I. (2) Either pTACR 102 B6 or pCR1001 was treated with Bam HI plus Kpn I to produce a DNA fragment comprising from Bam HI site (No. 15) of the prorennin cDNA base sequence up to KDn I site (No. 253). (3) pCR 1001 was treated with Kpn I plus Sal I to produce a DNA fragment comprising from Kpn I site (No. 254) of the prorennin cDNA up to the terminal Sal I site. Each DNA fragment obtained through steps (1), (2) and (3) was annealed together and, after annealing, the cyclic DNA pCR 2001 could be constructed by treatment with T4-DNA ligase. (6) Production of Prorennin by E. coli An aqueous medium (L broth) having the following composition was prepared: Ingredient Grams/liter Bacto-Tryptone (DIFCO) 10 Bacto Yeast extract (DIFCO) 5 Sodium chloride 10 pH 7.2 The strain, E. coli CR1 was cultivated at 370C under shaking in the neutrient medium containing 30 pg/ml of ampicillin, and lmM of IPTG (isopropylthio B-D-galactoside). Cells were harvested by centrifugation at 10,000 x g when propagation reached maximum. The Cells were then destroyed by sonication treatment and a cell-free extract was obtained. The prorennin protein in the extract was measured according to the radio-immunmoassay method using prorennin antibody labelled with 1251. As a result of the measurement, it was found that about 1,000 moleculars of prorennin protein were produced per one cell of the host microorganism. The extract was passed through a solid-state antibody column made of anti-prorennin Sepharose 4B, and the adsorbed protein was after elution tested on a SDSpolyacrylamide gel electrophenogram. The molecular weight of the product was thus confirmed to be similar to that of prorennin.

Claims: WHAT rs CLAIMS IS: 1. A recombinant plasmid comprising cDNA of calf prorennin. 2. A recombinant plasmid accoridng to claim 1 wherein the vector plasmid of said placid is pBR322. 3. A recombinant plasmid according to claim 1 which further comprises a promoter requisite for the expression of the prorennin producing gene in a microbial host cell. 4. A recombinant plasmid according to claim 3 wherein said promoter is the lac operon of E. coli and said host is E. coli. 5. Recombinant plasmid pCR1001 characterized by the fact it contains the complete base sequence of prorennin cDNA. 6. Recombinant plasmid pTACR102B6 characterized by the base sequence comprising from the first base at the N-terminus to the 890th base of prorennin cDNA. 7. Recombinant plasmid pTACR103C2 characterized by the base sequence comprising from the 771st base to the 1095th base at the C-terminus of prorenninn cDNA. 8. Recombinant plasmid pTACR5 characterized by the base sequence comprising from the base site before Kpn I site to the site after Bam HI site of prorennin cDNA. 9. Recombinant plasmid pCR2001 characterized by the fact it contains the prorennin cDNA sequence and the lac promoter region of E. coli lactose operon. 10. A microorganism containing a recombinant plasmid comprising cDNA of calf prorennin, which plasmid being inserted into a host microorganism. 11. A microorganism according to claim 10 wherein said host microorganism is Escherichia coli. 12. A microorganism according to claim 10 wherein said plasmid contains a promoter requisite for the expression of the prorennin producing gene in a host cell. 13. A microorganism according to claim 12 wherein said host microorganism is E. coli C600r m and said promoter is the lac operon of E. coli. 14. Microorganism E. coli CRl(rkmk, thr , leu thi pCR2001) characterized by the fact it contains plasmid pCR2001. 15. A process for making pCR1001 which comprises the steps of: (1) treating each of pTACR102B6 and pTACR103C2 with Sal I; (2) digesting the fragments from step (1) with exonuclease to produce fragments pTACR102B6' and pTACR103C2'; (3) treating fragment pTACR102B6' with Kpn I and fragment pTACR103C2' with Bam HI, respectively; (4) annealing the fragments from step (3) to produce fragment (a); (5) treating pTACR102B6 with Eco RI plus Kpn I to produce fragment (b); (6) treating large pTACR103C2 with Eco RI plus Bam HI produce to produce fragment (c); and (7) annealing fragments (a), (b), and (c) followed by linking by means of T4 DNA ligase. 16. A process for making pCR1001 which comprises the steps of: (1) treating pTACR102B6 with Hind III plus Kpn I to produce fragment (a); (2) treating pTACR5 with Kpn I plus Bam HI to produce fragment (b); (3) treating pTACR103C2 with Bam HI plus Hind III to produce fragment (c); and (4) annealing fragments (a), (b), and (c) followed by linking by means of T4 DNA ligase. 17. A process for producing calf prorennin which comprises cultivating in a nutrient medium, a genetically engineered microorganism which is constructed by incorporating a recombinant plasmid into a host microorgansim, said recombinant plasmid comprising cDNA of prorennin and a promoter requisite for the expression of the prorennin producing gene in a microbial host cell. 18. A process according to claim 17, wherein said host microorganism is E. coli and said promoter is the lac operon of E. coli. 19. A process according to claim 17 wherein said host microorganism is E. coli C600rkmk. 20. A process according to claim 17 wherein said recombinant plasmid is pCR2001. 21. A process according to claim 17 wherein said genetically engineered microorganism is E. coli CR1.

Inventors: BEPPU, TERUHIKO, NISHIMORI, KATSUHIKO, UOZUMI, TAKESHI
Applicants: BEPPU, TERUHIKO
Application year: 1982
Main classification: C12N15
IPC codes: C07H21, C12N15, C12N9, C12N1