DD264455A1 - PROCESS FOR SYNTHESIS OF CHYMOSINE - Google Patents

Abstract

The invention relates to a method by which the required for the production of cheese chymosin 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, so that in several stages a transformable plasmid is produced, which is transformed into a suitable Bacillus strain. After submerged and aerobic propagation of the host cells and lysis of the cell mass obtained, an activatable prochymosin can be obtained. Fig. 1

Description

For this 4 pages drawings

Field of application of the invention

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

Characteristic of the known state of the art

Chymosin, which is indispensable as a milk clotting enzyme, is derived from proteolytically inactive prochymosin, which in turn is isolated as is commonly known and commonly used in the abomasum of young ruminants. It is therefore available only to a limited extent, and its production in this way is cumbersome and expensive. It has therefore long been attempted to win prochymosin (or an equivalent pseudochymosin lacking some N-terminal amino acids without this deficiency being expressed negatively) by other means. It is obvious to synthesize the enzyme in a natural, but genetically modified microorganism, as has already been proposed in PCT application 8500382 and EP patent applications 133756 and 154351, for example, but which relates to the Daiegung a corresponding Limit tasks and describe the associated new procedure only in its basic features. This is not sufficient for carrying out the synthesis, because a productive secretion or the proteolytic degradation of chimeric, in the host host microorganism foreign proteins by its proteases is not readily, as already in "Proceedings of the National Academy of Sciences of the United States 79 (1982), pp. 5582-5586 It has indeed been attempted, according to PCT applications 8503949 and 8601825, to avoid the proteolysis of foreign proteins in the microorganisms used, and for the synthesis of prochymosin proposed method but not suitable and not provided.

Object of the invention

The invention aims to produce chymosin by an economic, largely synthetic process in any amount such that its properties match the natural on enzyme.

Explanation of the essence of the invention

The object of the invention is therefore to overcome the disadvantages of the known proposed methods and to synthesize a prochymosin which, in its chemical structure and its enzymatic properties, is exactly the same as the enzyme obtained from the abomasum. 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 modified in its region and in the region of the prochymosin sequence of the coding DNA.

According to the invention the object is achieved in that the N-terminal cONS sequence of the isolated donor DNA by acting before. Ligase is inserted into a promoter plasmid pAp8 (on a 475 bp BamHI fragment from a vector plasmid p 10c 10) such that a plasmid pCHY3 is formed such that the C-terminal cDNA sequence of the same donor DNA can be amplified by the action of ligase a KpnI-HindIII fragment from a vector plasmid p211) into the plasmid pCHY3 to form a plasmid pCHYKH2 and that the plasmid pCHYKH2 is formed into Bacillus in a known manner, conveniently into suitable Bacillus siibtilis 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.

embodiment

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

FIG. 1: the formation of a promoter piasmide pAp8, FIG. 2: the cDNS sequence of a natural gene for bovine procheine, FIG. 3: the formation of the plaswide pCHY3 and FIG

Fig. 4: the formation of the plasmid pCHYKH 2 with the complete from the N-terminal BamHI to the C-terminal cleavage site sequence of the prochymosin goose.

1. Preparation of promoter plasmid pAp8

Chromosomal DNA of a strain of Bacillus amyloliquefaciens, isolated according to the method of Saito and Miura (Saito, H., Miura, KI, Biochim., Biophys, Acta 72 M963J, pp. 619-629) and plasmid DNA of a vector plasmid pPL603 ( Williams, DM, Duvall, EJ and Lovoett, PS, J. Bacteriol 146 [1981], pp. 1162-1165) are digested by EcoRI in separate batches, deproteinized, ethanol precipitated, washed (Maniatis, T., Fritsch, EF and Sambrock, J., Molecular Cloning, A Laboratory Manual Cold Spring Harbor, NY, 1982) and with TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.5) so that a concentration of about 1 microgram per each 20 microliters of TE buffer is achieved. The EcoRI-digested chromosomal DNA (about 2 micrograms) and EcoRI-digested plasmid DNA (0.5 micrograms) are mixed in a 20 microliter assay in a reaction usual for T4 DNA ligase using about 2 units of enzyme (Maniatis, T. et al., Supra).

The ligation mixture is transformed into protoplasts of a Bacillus subtilis strain by a method of S.Chang and S.N. Cohen (Molec. Gen. Genet., 168 [19791, pp. 111-115]. The transformation of transformants is carried out on DM3 regeneration medium supplemented with 20 micrograms Cm / ml, 5 micrograms Km / ml and 1% insoluble starch. The transformant colonion are then separated on agar plates and checked for their Cm (chloramphenicol) degree of resistance. For example, out of 8 colonies, growth on nutrient agar plates at 200 micrograms / ml was still observed in one colony. The recombinant plasmid detectable and isolated from this transformant (eg, according to J. Bacteriol., 134 [1978], pp. 318-329) was designated pAp8 (FIG. This promoter plasmid differs from the plasmid pPL603 by an approximately 270 bp insert in the EcoRI site.

2. Preparation of plasmid pCHY3

Purified DNA from promoter plasmid pApB and a BamHI fragment above 474 bp with the N-terminal part of the calf prochymosin gene are required. The 474 bp fragment is obtained from a vector plasmid ρ 10c 10 (DD Pat. 'I36345; Folia biologica [Prahaj 31 [1985], pp. 71-85 and 81-92). For this purpose, approximately 10 micrograms of ρ 10c 10-DNA are digested in the first step under standard conditions using HindIII and Sail (in order to destroy the fragments of 1600 or 3300Bp which are unwanted for further cloning from the three BarnHI fragments resulting in the following step). In the second step, about 2 micrograms of DNA are digested under standard conditions using BamHI, deproteinizing the Hind III Sal I-digested DNA. By agarose gel electrophoresis, it is shown that the BamH I fragments (see above) which are undesirable for the subsequent ligation have actually been destroyed by the Hind IU-Sal I digestion carried out in the first step, ie three times (HindIII, Sal I, BamHI) digested ρ 10c 10 DNA is purified and is in ^-. TE buffer (about 8 micrograms per 10 microliters) is dissolved is available for ligation, under standard conditions Bcl I-digested about 1 microgram DNA of Promotorplasmids PAPB, subsequently deproteinised and the TE For ligation, 0.1 microgram of BclI-digested pApB DNA is ligated into 1 microgram HindIII-SalI-BamHI digested DNA of vector plasmid ρ10c10 under standard conditions. after (4h. at 21 ⁰ C) incubation of the ligation is terminated by heating to 65 ° C (for 5 minutes) and the transformation carried out in a Baciüus subtilis strain. in order to transform the above-mentioned method is nac h Chang and Cohen (1979). Transformants are selected on appropriate DM3 plates with addition of 750 micrograms / ml. The transformants obtained are checked by smear growth on nutrient agar for their Cm resistance; Thus, Cm-sensitive transformants are determined, hybridized by colony hybridization (Maniatis et al., 1982, Handbuch so) against a P-32-labeled prochymosin DNA-specific oligonucleotide Z3 GGTAACCCAGAAGTC and pre-selected after exposure of the hybridization filter to X-ray film as clones containing the N-terminal part of the prochymosin sequence. Of the thus preselectrinated Cm-sensitive transformants, plasmid DNA is isolated (for example, according to J. Bacteriul 134 (1978), pp. 318-329) and with the restrictases

EcoR I and Pst I are digested by standard methods to determine the location of the BamH I DNA fragment cloned in the gel I site of the vector. At desired orientation, the small of two expected fragments should be 301 bp (versus 631 bp in the reverse trap). The plasmid of some Trarisformantan always has the desired orientation of the BamH I DNA fragment (the promoter plasmid pAp8 In Flg. 3) on. Of these, plasmid pCHY 3 is selected for further operations, suitably propagated, isolated and provided as a purified DNA sample.

3. Preparation of plasmid PCHYKH2

Completion of the prochymosin gene (Figure 2) requires the steps illustrated in Figure 4 using the methods described in 2 above. They include a) the treatment of the DNA of plasmid pCHY3 by the standard method with the enzyme KpnI and Hind III, b) the isolation of a suitable Kpn I-Hind III DNA fragment of about 1000 bp by means of agarose gel electrophoresis and electro-elution except for the C-terminal prochymosin gene over about 940 bp from a vector plasmid p211 (Koebenik, R., "Expression of Prochymosin in Escherichia coli", Dipl.-Arb. 1986, Mart. Luther-Univ. Halle-Wittenberg) c) the ligation of Kpn I-Hind III-digested DNA of the vector pCHY3 and the Kpn I-Hind III DNA fragment by standard ligation procedures and d) the transformation according to Chang and Cohen, see 1.) in Bacillus subtilis GSB-26 (see under 2.) or into a Bacillus subtilis strain GB500 (DD patent 245444), so that tronsformant colonies are obtained, among which by colony hybridization (see 2. above) with P32-labeled synthetic oligonucleotides Z1 (homologous to the sequence TGATCAGATCGCTT in C Fig. 2) or Z3 (homologous to the sequence GGGTACCCAGAAGTC in the N-terminal part of the prochymosin sequence, Fig. 2)) are determined transformant colonies that give a positive hybridization response for the oligonucleotides Z1 and Z3 , Plasmid DNA is isolated from these transformants (for example, according to J. Eacteriol 134 (1978), pp. 318-329), after standard purification the DNA is isolated by means of restrictase digestions (preferably PstI, KpnI-Hind III, Ecorl. Hpal-Bglll) and electrophoretic separation in a 0.8% agarose gel and compared to the complete prochymosin DNA according to the plasmid restriction map expected in Figure 4. The plasmid corresponding to the expected restriction map is named pCHYKH 2.

4. Transformation into suitable host strains and evidence of prochymosin synthesis in Bacillus subtilis

The plasmid pCHYKH 2 thus obtained was transformed into the protease-defective Bacillus subtilis strain GB 500 (DD Pat. No. 2,454,444) using the Chang and Cohen protoplast transformation method (see 1. below). Plasmid pAp8 serves as a control after similar transformation into the Bacillus subtilis strain GB 500. After checking for the presence of plasmid pCHYKH 2 or pAp8 in the host cells of the bacilli subtilis strain GB 500, pure cultures of the transformants GB500-pCHYKH2 and GB500-pAp8 are incubated for 12.24 and 36 hours at 37 ° C. in a fermentation fermentor. Culture of Bacillus subtilis suitable nutrient medium (eg Na-succinate LGS according to Biochem Biophys Res. Commun., 128 (1985), pp. 601-606 or glutamate-casein medium according to Appl. Env. 'Microbiol., 50 (1985), Pp. 503-507). When the cells from these cultures are appropriately harvested and lysed, the cell extract can be analyzed by SDS-PAGE according to Laemmli (Nature 227 [1970], pp. 680-685) 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 Gruyter Berlin-New York) proposed casein-enzymogram technique or standard protein blottin (B. B. Proc. Nat. Acad. Sci., USA, 7611979), pp. 4350-4354) using chymosin-specific antibodies or by determining the milk-clotting activity (eg, according to Goff et al., Gene 27 [1984], p.35-46) prochymosin can be detected and determined.

Claims (4)

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 into a suitable genetic vector, for example a vector plasmid, and fused with a vector DNA to 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 by the action of ligase so (on a 475 bp Bam HI fragment from a vector plasmid ρ 10c 10) in a promoter plasmid pAp 8 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 Kpn I-Hind III fragment from a vector plasmid p211) in the plasmid pCHY 3, that a plasmid pCHYKH 2 is formed and - The plasmid pCHYKH 2 is transformed in a known manner in Bacillus. 2. The method according to claim 1, characterized in that the plasmid pCHYKH 2 is transformed into suitable Bacillus subtilis strains or other suitable microorganisms. 3. The method according to claim 1, characterized in that the transformed with dom plasmid pCHYKH 2 transformed host cells are suitably propagated submersed and aerobic. 4. The method according to claim 1 to 3, characterized in that the extracted Zeümasse is lysed in a suitable manner for fractionation of the prochymosin formed and therefrom activatable prochymosin is obtained.