DD253641A1 - PROCESS FOR SYNTHESIS OF CHYMOSINE - Google Patents

Abstract

The invention relates to a method by which the required for the production of cheese chymosin is no longer obtained from the abomasum of calves, but can be produced synthetically in any amount; at the same time, however, the synthesized natural chymosin should not differ in its chemical structure. For this purpose, an isolated chymosin gene is incorporated into a promoter plasmid, resulting in a multi-stage transformable plasmid, which is transformed into a suitable strain of Bacillus. After submerged and aerobic propagation of the host cells and lysis of the cell mass obtained, an activatable prochymosin can be obtained.

Description

Field of application of the invention

The invention relates to a process for the synthesis of chymosin.

Characteristic of the known technical solutions

The indispensable as a milk clotting enzyme chymosin is derived from the proteolytic ineffective prochymosin which ^ ie generally known and in use _(in turn isolated from the fourth stomach of young ruminants. Therefore, it is only to a limited extent available, and its production in this way is cumbersome and expensive It has long been attempted to otherwise obtain the prochymosin (or an equivalent pseudochymosin lacking some N-terminal amino acids without this deficiency being expressed negatively) .It is obvious that the enzyme is naturally occurring Genetically correspondingly modified microorganism to synthesize, as has already been proposed in PCT application 8500382 and EP patent applications 133756 and 154351, for example, but limited to the presentation of a corresponding task and describe the associated new method only in its basic features . The SI st is not sufficient for carrying out the synthesis, because a productive secretion or the proteolytic degradation of chimeric, in the host microorganism foreign proteins by its proteases is not readily carried out, as already in "Proceedings of the National Academy of Sciences of the USA" 79 (1982), pp. 5582-5586: It has indeed been attempted according to PCT applications 8503949 and 8601 825 to avoid the proteolysis of foreign proteins in the microorganisms used; for the synthesis of prochymosin, the proposed methods are not suitable and not intended.

Object of the invention

The object of the invention is to produce chymosin in an arbitrary amount by an economical, largely synthetic process in such a way that its properties are in accordance with the natural enzyme.

Explanation of the essence of the invention

The invention. Has therefore set itself the task of eliminating the above-mentioned disadvantages of the known proposed methods and to synthesize a prochymosin, which is exactly the same in its chemical structure and its enzymatic properties of the abomasum-derived enzyme. By secretion of the formed by a suitable microorganism prochymosin in a culture solution or by fractionation of the cell extract, the enzyme is to be produced as a filtered concentrate. In particular, a suitable secretion signal sequence must be selected and the primary structure in its region and in the region of the prochymosin sequence of the coding DNA modified.

According to the invention, the object is achieved in that the N-terminal cDNA sequence of the isolated donor DNA is incorporated by the action of ligase so (on a 475 bp Bam HI fragment from a vector plasmid p10c10) in a promoter plasmid pAp8 that a Plasmid pCHY3 results from the fact that the C-terminal cDNA sequence of the same donor DNA is incorporated into the plasmid pCHY 3 by the action of ligase (on a Smal-Hind HI fragment from a vector plasmid p8G8) in such a way that a plasmid pCHYA 7 is formed and that the plasmid pCHYA7 is transformed in a known manner into Bacillus, conveniently into suitable Bacillus subtilis strains. The thereby transformed host cells of Bacillus are suitably submerged and aerobic propagated, the lysed cell mass is lysed and therefrom activatable prochymosin is obtained. It is advantageous when plasmid pCHYA 7 is introduced into suitable Bacillus strains or other suitable microorganisms and checked for genetic stability and the formation of prochymosin with acid-activatable chymosin activity in order to produce production strains.

embodiment

The further details and advantages of the invention will be explained in more detail below with reference to the drawing aη an embodiment. Show it

1: the production of promoter plasmid pAp8 by in vitro recombination of EcoRI-digested vector plasmid pL603

FIG. 2: the restriction map of the cDNA of a natural gene for bovine prochymosin, FIG. 3: the formation of plasmid pCHY 3 with the N-terminal part of the prochymosin gene, and FIG. 4: the formation of plasmid pCHYA7 with the N-terminal BamHI site complete sequence of prochymosin gene.

1. Preparation of Promoter Plasmid pAp8

Chromosomal DNA of B. amyloliquefaciens ATCC15841, isolated according to the method of SAITO and MIURA (SAITO H., MIURA, K.-l .: Biochim.Biophys.Acta 72, 619-629, 1963), and plasmid DNA of the vector pPL603 (WILLIAMS , DM; DUVALL, EJ and LOVOETT, PS: J. Bacteriol., 146, 1162-1165, 1981) are digested by EcoRI in separate batches in a known manner, then deproteinized in a known manner, ethanol precipitated, washed (MANIATIS, T. FRITSCH, EF and SAMBROOK, J .; Molecular Cloning A. Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982) and dissolved with TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.5) so that a concentration of approx 1 ug per 20μΙ TE-buffer is achieved. The EcoRI-digested chromosomal DNA (about 2 μg) and EcoRI digested chromosomal DNA (about 2 μg) and EcoRI-digested plasmid DNA (0.5 μg / Ag) are 20 / xl in an approach for T4 DNA Ligase usual reaction using about 2 enzyme units (see MANIATIS ₁ T. et al., 1982, manual so) ligated.

The ligation mixture is transformed into protoplasts of B. subtilis strain BD170 (trpC2, thr5) by the method of S. CHANG and S.N.COHEN (Molec. Gen. Genet., 168, 11-115, 1979). The selection of the transformants is carried out on DM3 regeneration medium with the addition of 20 ^ g Cm / ml, 5Mg Km / ml, 1% insoluble starch. The transformant colonies are then separated on common culture plates and checked for their CrrHChloramphenicol-iResistenzgrades. For example, eight colonies were found to grow on nutrient agar plates at 200 μg / ml. The recombinant plasmid detected and isolated from this transformant by conventional methods (e.g., J. Bacteriol., 134, 318-329, 1978) was designated pAp8 (Figure 1). This plasmid differs from Vector pPL603 by an approximately 270 bp insert in the EcoRI site and is referred to as promoter plasmid pAp8.

2. Preparation of plasmid pCHY3

Required is purified by conventional methods purified DNA of promoter plasmid pAp8 and a BamHI DNA fragment of 474 bp with the N-terminal part of the gene for calf prochymosin. The 474 bp fragment is obtained from the vector plasmid p10c10 (DD patent 236345 and Folia biologica 31, 71-85, 1985). For this purpose, approximately 10 μg of piOdODNS are digested in the first step under standard conditions using HindIII and Sail (in order to destroy the fragments of the above-described three BamHI fragments which are unwanted for further cloning above 1,600 and 3300 bp). After customary deproteinization of HindIII / Sall-digested DNA, approximately 2 μg of DNA are digested under standard conditions using BamHI in the second step. By conventional agarose gel electrophoresis, it is shown that the BamHI fragments (see above) which are undesirable for the subsequent ligation have actually been destroyed by the HindIII / SalI digestion carried out in the first step. The three times (HindIII, Sail, BamHI) digested pi Od 0 DNA is purified by conventional methods and is in the TE buffer (about 8Mg per 10 / xl) available for disposal. For ligation, approximately 1 / Ag DNA of the promoter plasmid pAp8 is digested under standard Ball conditions, then deproteinized and dissolved in the TE buffer (to approximately 1 mg / 10 μl). For ligation, 0.1 μg / Bcll-digested pAp8 DNA and 1 / xg HindIII-SalI-BamHI-digested p10c10 DNA are ligated under standard conditions. After ligation (4h at 21 ⁰ C) this is terminated by heating to 65 ° C (for 5Minj and

used for transformation into B. subtilis strain GSB26 (metB5, SacA321, arol106, amyE, Str6 ^R ). For transformation, the method already mentioned above is used after CHANG and COHEN (1979). The transformants are selected on appropriate DM3 plates with the addition of 750 ^ g Km / ml. The transformants obtained are examined by smear growth on nutrient agar for their C _m resistance and 52 cm-sensitive transformants were determined. They are hybridized to a P ³² -labeled prochymosin DNA-specific oligonucleotide Z3 GGGTACCCAGAAGTC by standard colony hybridization (MANIATIS et al., 1982) and, after exposure of the hybridization filters to X-ray film, are preselected as clones containing the N-terminal portion of the prochymosin sequence , From the thus preselected Cm ^s transformants, plasmid DNA is isolated by conventional methods (eg, J. Bacteriol., 134, 318-329, 1978) and digested with the EcoRI and PstI restrictionases by standard methods to determine the location of the vector in the Bcll site of the Vector cloned BamHI DNA fragment. In the desired orientation, the small of two expected fragments 301 should be 631 bp in length (in the reverse case). The plasmid of some transformants usually has the desired orientation of the BamHI DNA fragment (to the promoter p8 in Figure 3). Of these, plasmid pCHY3 is selected for further operations, suitably propagated, isolated and provided as a purified DNA sample.

3. Preparation of Plasmid pCHYA 7

The second step required to complete the prochymosin gene (FIG. 2) involves the steps illustrated in FIG. 4 using the methods described under 2. They include a) the treatment of DNA of plasmid pCHY3 by the standard method with the enzymes SmaI and HindIII, b) the isolation of a suitable SmaI-HindIII DNA fragment by means of agarose gel electrophoresis and electroelution (pages 124-124 in RF Schlenk and PCWensink, Practical methods in molecular biology, Springer-Verlag, 1981) with about 1440 bp, which also contains total C-terminal Prochymosingen over 779 bp from the vector plasmid p8G6 (DD patent 236345), c) the ligation of Smai-HindIII digested DNA of Vector pCHY 3 and SmaI-HindIII DNA fragment above about 6440 bp by standard ligation method (at about 10-fold increased T4 DNA ligase concentration to improve blunf end ligation compared to sticky / end ligation in section 2) ) and d) the transformation of the ligated DNA using the protoplast transformation according to CHANG and COHEN (see Section 1) into the B.subtilis strain GSB-26 (see Section 2), so that transformant colonies he under which by using the usual Koloniehybridisierungsmethode (s. Section 2) by hybridization with P ³² -labeled synthetic oligonucleotides Z1 homologous to the sequence TGATCAGATCGCTTT in the C-terminal part of the prochymosin sequence (FIG. 2) or Z3 (homologous to the sequence GGGTAGCCAGAAGTC in the N-terminal part of the prochymosin sequence (FIG ) the transformant colonies 7 and 9 are determined, which give a positive hybridization response both for the oligonucleotide ZI and Z3. From these, for example, two transformants, plasmid DNA is prepared by a conventional method (eg J. Bacteriol 134, 318-329, 1978) isolated and after standard purification of the DNA by means of Restraktaseverdauungen (preferably SmaImal, SmaI-HindIII, Bllll-HindIII, Bell, Hpal and Bglll) and conventional electrophoretic separation in a 0.8% agarose gel identified and with those of Figure 4 in agreement The plasmid of a transformant colony corresponded to the expected Res and receives the designation pCHYA7.

4. Transformation into suitable host strains and detection of prochymosin synthesis in B. subtilis

The resulting plasmid pCHYA 7 is transformed into the protease-defective B. subtilis strain GB500 (patent DD 245444) using the protoplast transformation method according to CHANG and COHEN (see Section 1). Plasmid pAp8 serves as a control after similar transformation into GB500. After checking for the presence of plasmid pCHYA7 or pAp8 in the host cells of strain GB500 (using standard plasmid isolation techniques), pure cultures of transformants GB500-pCHYA7 and GB500-pAp8 are incubated for 12.24 and 36 h at 37 ° C in a fermenter culture of B. subtilis suitable nutrient medium (eg Na succinate LGS Biochem Biophys Res Commun 128, 601-606, 1985 or glutamate-casein medium according to Appl.Env Microbiol 50, 503-507, 1985 ) carried out. When the cells from these cultures are appropriately harvested and lysed, cell extract can be analyzed by SDS-PAGE according to LAEMMLI (Nature 227, 680-685, 1970) using the methods described by FOLTMANN-et al. (in FEBS Advanced Course on aspartic proteinases and their inhibitors, Prague 20-24.10.1984, published by Walter de Gruyter Berlin-New York) proposed casein enzymogram technique or by standard protein blotting (eg Proc. Nat. Acad. 4350-4354, 1979) can be detected and determined using chymosin-specific antibodies or by determining the milk-clotting activity (eg, according to GOFF et al., Gene 27, 35-46, 1984).

Claims (5)

claims: 1. A method for the synthesis of chymosin, wherein A genetic substance capable of synthesising prochymosin is isolated from a natural animal organ, such as a calf's stomach, - a DNA fragment is made from the genetic material that is useful as donor DNA, The donor DNA is incorporated in a suitable genetic vector, for example a plasmid, and fused with a vector DNA to form a DNA chimera molecule, The DNA chimera molecule is transformed for the purpose of its propagation into suitable host cells, preferably microorganisms of the genus Bacillus, and Finally, from the enzyme produced intracellularly by the host cells, by fractionation of the cell extracts, prochymosin is finally obtained, characterized in that The N-terminal cDNA sequence of the isolated donor DNA is incorporated into a promoter plasmid pAp 8 by the action of ligase (on a 475 bp Bam HI fragment from a vector plasmid p10c10) in such a way that a plasmid pCHY 3 is formed, - The C-terminal cDNA sequence of the same donor DNA by the action of ligase is so incorporated (on a Sma! -Hind III fragment of a vector plasmid p8G8) in the plasmid pCHY3 that a plasmid pCHYA 7 is formed and - The plasmid pCHYA 7 is transformed in a known manner in Bacillus. 2. The method according to claim 1, characterized in that the plasmid pCHYA7 is transformed into suitable Bacillus subtilis strains. 3. The method according to claim 1, characterized in that the transformed with the plasmid pCHYA7 host cells of Bacillus are suitably propagated submersed and aerobic. 4. The method according to claim 1 to 3, characterized in that the cell mass obtained is lysed in a suitable manner for fractionation of the prochymosin formed and therefrom activatable prochymosin is obtained. 5. The method according to claim 1, characterized in that for the production of production strains plasmid pCHYA 7 are introduced into suitable Bacillus strains or other suitable microorganisms and checked for genetic stability and the formation of prochymosin with acid-activatable chymosin activity out. For this 4 pages drawings