CS270703B1 - Method of foreign proteins' secretory production by means of transformed yeast cells cultivation - Google Patents

Abstract

The present invention relates to the preparation of foreign proteins modified by the yeast S. cerevisiae cell genetic engineering procedures. Cells yeast transformed with zwitterionic derivatives the pJDBa vector containing the coding combination sequences with the yeast and phak gene -MFa, provide high con- Centering the protein product of the introduced gene. The pJDBoc vector derivatives are designated pJDBaCH and pJDBaCHE, contain the gene sequence for the Prochymosin attached to the correct signal MFa sequence. Designed transforming vector solves the general connection method a mature coding sequence foreign protein, using protease the KEX2 yeast host cell system performs a translational translational release adjustment producing protein from the primary translation product.

Description

The invention relates to a method for the secretory production of foreign proteins by a yeast cell. Cells of prokaryotic organisms Escherichia coll and Bacillus subtilis and eukaryotic cells of the yeast Saccharomyces cerevisiae are most commonly used for the production of foreign proteins. In order for the foreign gene encoding the protein to be maintained and expressed in the new host, it is necessary to construct suitable helper DNA molecules, so-called expression vectors, into which the foreign gene is inserted in vitro using recombinant DNA technology. Vector DNA molecules contain a number of sequences that facilitate the selection of cells into which the vector, resp. Recombinant DNAs entered (transformed cells), replicating these DNAs. in one or more hosts (shuttle) vectors) and the transcription or translation of the inserted foreign region in the new host, respectively. Recently, vectors that provide not only the expression of a foreign gene or a portion thereof, but also the secretion of a functional protein product or its precursor into the periplasmic space or even into the culture medium have been particularly appreciated. In these cases, there is no need to isolate and complicate the purification of the foreign protein from within the host cell. Particularly suitable host organisms are S. yerevisiae yeast cells, in which the number of intrinsic proteins produced in the medium is relatively small. which facilitates the purification of the foreign protein from the medium. A number of yeast genes have been described in the literature, the introductory, so-called signal sequences of which can be used to ensure the secretion of a foreign protein. Among them, the particularly well-studied MF «gene, which encodes the sexual yeast teromone, the so-called α-factor, plays an important role, see Fig. 1. This gene has become an important part of various vectors described in the literature that provide not only expression but also secretion of foreign proteins from the yeast S. cerevisiae (Bitter et al., Proc, Natl. Acad. Sci. USA 81: 5330, 1984).

The essence of the method of secretory production of foreign proteins by the yeast cell Saccharomyces cerevisiae according to the invention is that the yeast cell is transformed with derivatives of the zvD vector pJDB carrying a combination of coding sequences with the yeast α-factor -MFa gene.

- sequences ensuring the replication of the vector in Escherichia coll,

- sequences ensuring the replication of the vector in the yeast S. cerevisiae,

- sequences ensuring the selection of transformed E. coli clones based on amp ^r a

- a sequence ensuring the selection of transformed S. cerevisiae leu2 ~ clones based on the complementation of the leu2 mutation.

The presence of unique cloning sites within the MFα gene allows the attachment of a foreign coding sequence downstream of the MFα secretion signal sequence. In addition, the structure of the pJDBa vector containing the sequence encoding the amino acids recognized by the KEX2 protease system of S. cerevisiae allows the use of this system to ensure the secretion of the mature protein into the medium.

Derivatives of the pJDBCt vector are characterized by the content of foreign coding sequences inserted into the MFa factor of the gene in the pJDBa vector. an MFα signal sequence that provides for the expression and secretion of functional chymosin in a S. cerevisiae yeast cell. Thus transformed S. cerevisiae cells produce and secrete functional chymosin into the medium.

To construct the pJDB «C vector, MFct sequences were obtained as part of plasmid p69A (Kurjan and Herskowitz, Cell 30; 933), which is not itself useful as an expression-secretion vector because it does not contain unique sites for cloning foreign genes, see Figure 2. said coding sequences contain a useful fragment of this plasmid as well as promoter and terminator regulatory sequences and adjacent yeast chromosomal regions. All the sequences required for the construction of the expression-secretion vector lie in the plasmid between the two EcoRI target sites, see Figure 2.

From the restriction map, see Fig. 2, and the complete sequence of part of this region, see Fig. 1, it follows that at the beginning of each copy of Qt-factor is a HindIII site that can be used to attach a foreign gene to the MF signal secretion sequence. Insertion in the required orientation can be achieved by substituting the foreign gene for the HindIII-SalI fragment of MFa. However, because plasmid p69A contains a larger number

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Hind III and SalI sites outside the "ol" segment and their removal would be quite difficult, it was more advantageous to transfer MRt to another commuting vector - pJDB2o7 (Beggs, Molecular genetics in the east, Alfred Benzon Symp. 16: 383, 1981) - from which the Hind III and Sall sites were removed in advance. The construction scheme of such a plasmid pJDBd, in which the plasmid pBR322 was also used, is shown in Fig. 2. E. coli cells transformed with the plasmid pJDBd were selected on the basis of amp ^r ,

According to the invention, an EcoRI fragment carrying MFα was inserted into plasmid pJDBa in several ways, but only insertion into the EcoRI site outside the plasmid LEU2 gene provided two suitable resulting pJDBα vectors with different MFα orientation. Restriction mapping and expression of the amp ^r , LEU2 and MFd genes in E. coli and S. cere vis iae were used to analyze the resulting plasmids.

The pJDBa vector consists of double-stranded circular DNA with a contour length of about 7.4 kbp. It consists of these main functional parts, see Fig. 2 t

a) A DNA fragment containing ori (origin of replication) from the ColE1 plasmid, which ensures the replication of the vector and its derivatives in Escherichia coli. Since ColE1 is a plasmid with a relaxed form of replication, the pJDBa vector (or its derivatives) can be amplified in bacterial exciters after the addition of chloramphenicol and isolated in sufficient quantities.

b) A DNA fragment containing the bacterial amp ^r gene, derived from plasmid pBR322, which confers resistance to the antibiotic ampicillin antibiotic on transformed E. coli and provides the possibility to select transformants. The transformation efficiency of E. coli HBlol (Maniatis et al., 1982) with this vector is orders of magnitude comparable to the transformation efficiency of other commuting vectors (E. coli, S. cerevisiae) derived from plasmid pBR322.

c) A 2 yU DNA fragment (form B) from S. cerevisiae containing ars (abbreviation derived from the term "autonomously replicating sequence"), which ensures replication of the vector and its derivative in a number of yeast cell species (eg S. cerevisiae, Schizosaccharomyces pombe and Kluyveromyces lactis). '

d) The LEU2 gene from S. cerevisiae, which has an attenuated promoter so that fewer copies of the respective mRNA are produced in S. cerevisiae, and thus the production of a functional enzyme belonging to the leucine synthesis metabolic pathway is reduced compared to the LEU2 gene equipped with normal promoter. When the S. cerevisiae leu2 mutant, which cannot grow on minimal medium without the addition of leucine, is transformed with the pJDBa vector or its derivatives, the transformant acquires the ability to grow on this medium. Due to the attenuated expression of the LEU2 gene, the selection pressure increases the copy number of the vector in transformants growing on minimal medium, as a low copy number of the vector does not provide a sufficient amount of leucine. Thus, the LEU2 gene contributes both to maintaining a high copy number of the pJDBa vector in transformed cells growing on minimal medium and to the relatively good stability of the vector within the population, see Example 1.

e) DNA fragment from S. cerevisiae containing the MFa gene including promoter and terminator, see Fig. 1. These sequences ensure the production of d-factor from transformed S. cerevisiae feather-type cells and into the medium and can be used to secrete a foreign protein encoded by an exogenous sequence. , which can be inserted into the MFα gene in an appropriate manner to replace part of its original sequence. The distribution of restriction sites is shown in Figures 1 and 2. Hind III and Salt restriction sites are particularly important for the construction of recombinant DNA derivatives of the pJDBa vector that can provide secretion of foreign protein, allowing certain sections of the vector to be exchanged for recombinant DNA technology methods described in the literature (e.g., Maniatis et al., Molecular Cloning, A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982),

To ensure the expression and secretion of calf chymosin (rennet) by a transformed S. cerevlsiae cell, a prochymoal gene from plasmid p424, flanked by HindIII and SalI sites, regardless of the translation phase - pJDBdCH, was inserted into the pJDBa vector in this way, see Figure 3. In the second step modification of this plasmid was performed, which shifted the prochymosin sequence to the correct translation phase - pJDBaCHE, see Fig. 3, which could be analyzed in the absence of HindIII.

CS 27o7o3 Bl places. Both vectors - pJDBACH and pJDBdCHE - were further characterized by a trick with Sm cleavage (EcoR1, SalI, PstI, BamHI, SmaI), see Fig. 3, and confirmation of the function of all important gene sequences (ori, amp ^r , LEU2 and 2 yU ars ) in E. coli or S. cerevisiae hosts.

S. cerevisiae strain GRF18 («his3 ~ leu2 ~) was used as a host strain for transformation with the prepared vector pJDB & CHE. Transformation was performed either at the level of protoplasts in the presence of CaCl 2 and PEG 400 (modified according to Xin-Liang Zhu et al. Mol. Gen. Genet. 194 t 31, 1984), or competent cells prepared by LICI (Rodriguez and Tait, Recombinant DNA Techniques : An Introduction, The Benjamin / Cummings Publishing Company. Inc., London, Amsterdam, Don Mills Ontario, Sydney, Tokyo, 1983).

In several clones transformed with protoplast. resp. The lid method was used to determine ploidy by measuring the volume distribution of cells in the population of a given clone on a Coulter Counter. The LIC transformants were, as expected, haploid in all cases, while the protoplast transformants were diploid in many cases, thus apparently formed by fusion during transformation.

In this example, transformation of the GRF18 strain with the vector pJDBcCCHE showed that the expression-secretion vector pJDBa, resp. its derivatives, both protoplasts and intact yeast cells can be transformed to obtain transformants with different ploidy.

pJDBac /Ε / G RF18 transformants were tested for their ability to produce functional chymosin using a milk-clotting assay.

A buffer in which pJDBCCCHE-transformed S. cerevisiae GRF18 cells grown on an agar plate were pre-resuspended, as well as a liquid post-culture medium, see Table 2. GRF18 clones transformed with pJDBaCH or pJDBa and the parental GRF18 strain were used as controls. recorded only when buffer or medium was used after culturing the pJDBaCHE strain or a calf chymosin control preparation (Serve) and confirms not only the expression of the prochymosin gene using the vector-hostile system but also the secretion of the gene product into the medium as active enzyme. This result is also confirmed by the inhibition of the clotting activity by pepstatin (an aspartate inhibitor for teas), while the PMSF (phenylmethylsulphonyl fluoride inhibitor of serine proteases) used in the parallel test was ineffective,

Thus, only a strain that has been transformed with a plasmid containing the prochymosin gene in the correct translational phase downstream of the MFa preprosequence produces a substance with activity in a milk-clotiing assay. This activity, like the activity of a commercial chymosin preparation, is inhibitable by pepstatin or is not inhibitable by PMSF. Because neither the original parent strain GRF18 nor GRF18 transformed with the vector pJDBa, resp. pJDBaCH, which contains a prochymosin gene inserted in the wrong translation phase, does not show similar activity, it can be assumed that this active substance is chymosin produced in the medium as an active enzyme.

The following are examples of the construction of secretory-expression plasmids, the production of chymosin in culture medium, and the use of plasmids pJDBa for the general secretion of a foreign protein product into the medium.

Example 1

Construction of the secretion-expression vector pJDBd

Construction of pJDBif vector (Fig. 2)

The vector pJDB2o7 (ca. lo yUg) was digested with the restriction endonuclease HindIII (lo units | Maniatis et al., Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). The cohesive 5 ends of about 5 .mu.g of digested plasmid DNA were supplemented with a Klenow fragment of DNA polymerase 1 (5 units). After the subsequent cleavage of Pst! (5 units), the DNA sample was separated by electrophoresis (0.8% agarose gel) and a larger fragment carrying LEU2, 2yU are, and a portion of the amp ^r was purified by electroelution from the excised portion of the gel. The vector pBR322 (ca. lo yUg) was digested with PstI (lo units) and PvuII (lo units). The DNA was electrophoretically separated (0.8% agarose gel) and the smaller fragment containing ori and part of amp ^r was

CS 27o7o3 B1 purified again by electroelution. The size of the purified fragments determined electrophoretically in an .8% agarose gel corresponded to the predicted dimensions derived from the restriction maps of the starting plasmids pBR322 and pJDB20. The fragments thus obtained (ca. ng were ligated with T4-DNA ligase (ca. 1.5 Units) in a reaction volume at 26 DEG C. for 6 h. Competent E. coli HBlol cells were transformed with the ligation mixture (ca. 50 ng) and seeded on ZA (4% nutrient agar; Saris Michalany) containing 50 ug ampicillin / ml From individual amp ^r clones grown in 1 ml ŽB-amp medium (2.5% nutrient broth; Saris Michalany; containing 50 ug ampicillin / ml) via Plasmid DNA was isolated in a thermostat overnight at 37 DEG C. (Holmes and Quigley, Anal. Biochem. 114: 193, 1981), and the size of the plasmid DNA molecules was compared electrophoretically in an 8% agarose gel with the size of the starting plasmid pJDB20. DNA smaller than pJDB2o7 was further characterized using the restriction endonucleases EcoRI, PstI, Hind III, SalI and BamHL

The putative pJDBtT construct differs from the starting plasmid pJDB20o as follows:

a) does not contain HindIII, SalI and BamHI restriction sites

b) is generally smaller by about 1.45 kbp

c) contains only 2 EcoRI sites (within the LEU2 gene and between LEU2 and amp ¹ genes)

d) a large EcoRI fragment. pJDBd is larger than the largest fragment of pJDB20o7

Verification of the function of marker genes on plasmids pJDB;

a) Transformed E. coll cells (transformation method used - Maniatie et al., Molecular cloning · A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 25o, 1982) are resistant to ampicillin (5o yUg / ml), confirming the presence of the amp ^r gene and a functional ori.

b) Plasmid pJDB <T transforms S. cerevisiae GRF18 (ft hls3 ”ieu2 '*) with the same efficiency as plasmid pJDB20? (transformation method used - Xin Liang Zhu et al., Mol. Gen. Genet. 194; 31, 1984), which indicates the presence of a functional 2yU ars.

c) Transformed cells of the auxotrophic strain GRF18 (ft his3 ~ leu2 ⁺ ) are grown on minimal agar medium (1% glucose, 0.1% KH ₂ PO ₄ , 0.1% MgSO _4> 0.5% (NH 4 SC 2, 0.1% Wickerham solution of vitamins (Maráz and Ferenczy, Protoplasta - applications In microbial genetics, Ed. Peberdy, JF, Nottingham · 34, 1979), 1.5% agar) without leucine, indicating the presence of a functional LEU2 gene in pJDBd Transformed cells do not grow on minimal medium without histidine.

Construction of vector pJDBft. (Fig. 2)

The vector pJDB <γ (ca. lo yUg) was partially digested with EcoRI (lo units; Maniatis et al., Molecular Cloning · A laboratory manual · Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982), DNA was electrophoretically separated. The 8% agarose gel and the linear form of the vector were isolated by electroelution from the gel. The p69A vector (ca. lo yUg) was digested with EcoRI (lo units) and after electrophoresis, a fragment of the size corresponding to the EcoRI fragment containing the complete MF «gene including promoter and terminator sequences (ca. 1.6 kbp) was also purified by electroelution from an 8% agarose gel. see Fig. 2. A fragment representing the linearized vector pJDB <f (ca. 50 ng) and an EcoRI fragment containing the MFft gene (ca. loo ng) were ligated with T4-DNA ligase (0.1 Units) in a reaction volume of louul at 15 ° C 5 h. Competent E. coli HBlol cells were transformed with the ligation mixture (ca. 50 ng) and transformants were selected based on ampicillin resistance (50 ug / ml) on ŽA-amp medium. Plasmid DNA (Holmes and Quigley, Anal. Biochem. 114 t 193, 1981) was isolated from individual transformed clones grown overnight in 1 ml of ŽB-amp medium at 37 ° C in a thermostat and compared to the size of the vector on an electrophoretic gel. pJDBď. Those isolates that contained plasmid DNA larger than the pJDBtf vector were further characterized using the restriction endonucleases EcoRI, PstI, HindIII and SalI. Recombination of the starting fragments in vitro could result in 4 basic types of recombinant plasmids, 2 of which are suitable for the target of the construct. Suitable constructs could be easily selected

CS 27o7o3 B1 based on the transformation of S. cervisiae GRF18 (Xin-Liang Zhu et al. ₍ Mol. Gen, Genet. 194: 31, 1984)), only the vector that contained the MFé gene between the LEU2 gene and amp ^r carried a functional LEU2 gene, which complemented after transformation of the host strain S. cerevisiae GRF18 (Λ his3 leu2 ~) Its leu2 mutant allele Both types of orientations could be distinguished by PstI restriction digestion. in the orientation shown in Fig. 2. In this case, PstI fragments of medium size are not formed, but only a large fragment of about 5.9 kbp and two small fragments of about 0.8 and 0.7 kbp. ).

The function of all important sequences on plasmid pJDBA was verified and confirmed; ori and amp ^r by E. coli transformation; LEU2 and 2yU ars by S. cerevisiae transformation.

In addition, GRF18 transformants were tested for factor overproduction by a zonal assay, which is based on the fact that the growth of S. cerevisiae feather-type cells is inhibited by β-factor produced by type A cells. The zonal assay was performed as follows: 0.1 ml of stationary suspension S. cerevisiae AH215 cells (and his3 ”leu2 *) were plated on the surface of a minimal agar plate with histidine and leucine (50 yug / ml). Test clones of S. cerevisiae GRF18 were further plated and the plates were incubated at 30 ° C for 3 to 4 days. An inhibition zone appeared around the AH215 substrate around the GRF18 clones transformed with the pJDB vector, indicating an overproduction of d-factor by these clones. No detectable zone was detected around the parent strain GRF18.

Stability of pJDBOI vector:

The stability of the expression-secretion vector pJDBd in the host strain S. cerevisiae GRF18 was tested as follows:

Colonies of S. cerevisiae GRF18 / pJDB grown on selective minimal agar medium with histidine (50 yug / ml) (MA-his) were resuspended in distilled water and plated on non-selective minimal agar medium with histidine and leucine (50 yug / ml) (MA -his, leu). Individual colonies were picked, resuspended in distilled water, cell counts were determined by cell counting, and the undiluted suspension was resuspended in MA-hisJeu medium. Individual colonies were tested for their ability to grow on MA-his in the absence of leucine. The fraction of colonies that did not grow on MA-his medium was about 7%.

Since there are about 10 cells per colony that arise from a single cell in about 20 generations, it can be concluded that in 2 generations, about 7% of the cells lose vector DNA.

Example 2

Cloning of the calf prochymosin gene cDNA and construction of plasmid derivatives pJDBaCH and pJDBOLCHE

The gene for calf prochymosin was available as part of plasmid p424 (Sedlacek et al., Folia Biologica (Prague), 33: 145, 1987), in which it is inserted from the fifth amino acid into the polylinker HindIII-PstI-SalI-BamHI - prochem-PstI -SalI-BamHI-SmaI-EcoRI in the BamHI-PstI orientation, see Fig. 3. The prochymosin gene could be inserted into the pJDBd vector by substitution. in exchange for a Hind III-Sall fragment. However, it follows from the sequences of both genes that the prochymosin gene thus inserted is not in the correct translational phase with the secretory sequence MF, see Fig. 3. Therefore, the construction procedure had to be divided into two steps.

In the first, the pJDBd vector was recombined in vitro, from which a short section was excised with HindIII and SalI (ca. 5 μg of pJDBO DNA vector, digested with 5 HindIII and SalI units; Maniatis et al., Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982) with the prochymosin fragment of HindHI-Sall prepared as follows:

AND

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a) plasmid p424 (ca. lo / Ug) was first partially digested with SalI (lo units) and the linear form was isolated from the gel by electroelution after electrophoretic separation (0.8% agaroe gel),

b) The isolated fragments were digested with HindIII (ca. 5 units), separated by electrophoresis in an 8% agarose gel, and the HindIII-Sail fragment carrying the prochymosin gene was isolated again by electroelution. Vector of pJDBa. The digested HindIII and SalI (ca. 50 ng) and the HindIII-SalI fragment containing the prochymosin gene (ca. 10 ng) were ligated with T4-DNA ilgas (0.1 l units) at 15 ° C for 6 h. The ligation mixture (ca. 50 ng) was transformed E. coli HBlol and transformants were selected on the basis of amp ^r on ŽA-amp medium. Plasmids (Holmes and Quigley, AnaL Biochem. 114, 193, 1981) were isolated from individual transformed clones grown overnight in FIB-imp medium at 37 ° C in a thermostat and their size was compared electrophoretically with the size of the starting vector pJDBa. Plasmids larger than the pJDBa vector were further characterized by digestion with the restriction endonucleases EcoRI, HindIII, PstI, SalI, SmaI, and BamHL. differs as follows;

a) additionally contains SmaI, 3 BatnHI, 1 EcoRI, 1 SalI and 2 PstI sites inside the prochymosin fragment

b) is generally larger by about 1.13 kbp

c) After SalI digestion, a fragment containing the prochymosin gene corresponding in size to the Salt-HindIII prochymosin fragment used for cloning was excised from the pJDBaCH vector. The second fragment after digestion of SalI corresponds in size to the linear form of the starting plasmid pJDBa.

d) In addition, after cleavage of PstI, a fragment almost similar in size to the HindIII-SalI prochymosin fragment is formed (87 bp shorter).

In a second step, the amplified vector pJDBaCH was isolated from E. coli (Maniatia et al., Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982) and the phase shift was performed as follows with pJDBfitCH (ca. 1 .mu.g) was linearized with HindIII (1 unit) and the cohesive 5 'ends were supplemented in vitro with a Klenow fragment of DNA polymerase 1 (1 unit). Ligation of about 30 ng of plasmid DNA thus prepared by T4-DNA ligase (1.5 units) took place in a reaction volume of 1 yul at 26 ° C for 8 h. E. coli cells were transformed with the ligation mixture (about 50 ng) and smp ^r clones on ŽA-amp medium. Plasmids (Holmes and Quigley, Anal. Biochem. 114-193, 1981) were isolated from individual clones grown overnight in ŽA-amp medium at 37 ° C in a thermostat and digested with the restriction endonuclease HindIII. After electrophoretic analysis, three isolates were selected that did not linearize plasmid DNA by HindIII, which did not contain a HindIII restriction site - pJDBaCHE. Their structure was further verified by restriction digestion (EcoRI, SalI, PstI, BamHI, SmaI). The resulting pJDBa C HE vector was restricted to the pJDBccCH vector only in the absence of a HindIII site.

Furthermore, the function of all important sequences on the vectors -pJDBaCH and pJDBotCHE was checked and confirmed; ori, amp ¹ , LEU2 and 2 / U ars in both E. coli and S. cerevislae hosts, see Example 1.

Example 3

Transformation of S. cerevisiae with the recombinant vector pJDBaCHE and secretion production of calf chymosin.

The S. cerevisiae strain was used as the host strain for transformation with the pJDBaCHE vector

GRFlfl (a his3 ~ leu2 ^- ). The transformation was performed in two ways:

a) Transformation of GRF18 protoplasts in the presence of ^CaCl ₂ O 2 PEG 400 in a modified manner according to Xin-Liang Zhu et al., (Mol. Cen. Genet. 194, 31, 1984).

The culture of S. cerevisiae GRF18 grown to exponential phase in YEG culture medium (1% glucose, 0.5% yeast extract) was centrifuged, washed with distilled H ₂ O and incubated

CS 27o7o3 Bl for 15 min in 1% β-mercaptoethanol / HgO at room temperature. The cells were further washed with 1M sorbitol and incubated in freshly prepared zymolysis solution (1 mg zymolyase / ml ml 1M sorbitol). Control of protoplast formation was continuously performed microscopically. The protoplasts were washed with 1M sorbitol and STC solution (1M sorbitol, 10 mM Tris-HCl pH 7.5, 10 mM CaCl 2) and finally resuspended in STC to a final concentration of about 10 protoplasts / ml. Approximately 5 [mu] g of plasmid DNA was added to 200 [mu] l of protoplast suspension in STC and, after 15 min incubation at room temperature, 2 ml of PTC solution (40% PEG4000, 10 mM Tris-HCl pH 7.5, 10 mM CaCl2). The mixture was incubated for 30 minutes at room temperature, centrifuged and the sedimented protoplasts resuspended in STC. The variously undiluted transformation mixture, to which regeneration agar (3% agar, 0.3 M CaCl 2) warmed to 45 ° C was added, was seeded. substrate of selective osmotically stabilized (0.6 M KCl) minimal agar medium containing histidine (So yug / ml) and cultured at 30 ° C for 7 to 9 days. Histidine was tested for auxotrophy in grown clones. The frequency of transformation was on average lo- ⁴ transformants.

b) Transformation of competent GRF18 cells prepared with LiCl (Rodriguez and Tait, Recombinant DNA Techniques: An Introduction, The Benjamin / Cummings Publishing Company, Inc., London, Amsterdam, Don Mills Ontario, Sydney, Tokyo, 1983).

The culture of S. cerevisiae GRF18 grown in YEG medium to ODG _4o »2 1.5 was centrifuged, resuspended in TE buffer (lo mM Tris-HCl pH 7.5, about l mM EDTA), & again centrifuged. The cells were resuspended in LA solution (0.1 M lithium acetate, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and incubated aerobically for 60 min at 30 ° C. After centrifugation, the cells were resuspended in LAG solution (0.1 M lithium acetate, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 15% glycerol) to a final concentration of 2 to 5 μl cells / ml. To 0.3 ml of the suspension was added 5 to 10 .mu.g of vector DNA, 0.7 ml of 50% PEG40,000, and the mixed mixture was incubated for 60 minutes at 30 ° C and 5 minutes at 42 ° C. The transformation mixture was then plated on the surface of selective minimal agar medium with histidine (5 μg / ml) and incubated at 30 ° C for about 7 days. The grown clones were tested for histidine auxotrophy. The frequency of transformation was on average lo ?.

pJDB (tCH / GRF18 transformants were tested for their ability to produce functional chymosin using a milk-clotting assay. Standard performance of the milk-dotting assay:

To 5 [mu] l of milk suspension (25 mg of defatted Eliga dissolved in 0.1 M CaCl2) was added 5 [mu] l of variously diluted test sample and incubated for 2 h at 37 [deg.] C., the non-coagulated milk was removed with filter paper. In a control experiment with a chymosin preparation (Serva), it was confirmed that, in accordance with the literature data (Mellor et al., Gene 24 and 1, 1983), clot formation still occurs in a sample containing about 0.3 .mu.g of chymosin.

A buffer in which pJDBitCHE-transformed S. cerevisiae GRF18 cells grown on an agar plate were pre-resuspended, as well as a liquid post-culture medium, see Table 2. GRF18 clones transformed with pJDBaCH or pJDBct and the parent GRF18 strain were used as controls. A positive result was recorded only when buffer or medium was used after culturing the pJDB CHE strain or a calf chymosin control preparation (Serva), see Table 1.

with

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Table 1

Results of the milk-clotting test

solid medium liquid medium ... his MA—, leu wort MM-CA YEPG GRF18 - - - pJDBa / GRF18 - - • - pJDBctCH / GRF18 - - - - pJDB «.CH / GRF18 + + 4 · +

MA minimal agar medium with 1% glucose and histidine (5o yug / ml), in case?

GRF18 strains additionally with leucine (5o yUg / ml)

MM — CA minimal medium with 2% glucose and casamino acids (0.5%)

YEPG complex medium (2% glucose, 1% yeast extract, 1% peptone)

Test clones were removed from individual plates and resuspended in 50 μl TM buffer (10 mM Tris-Cl, pH 6.5, 10 mM MgCl 2). After centrifugation, 5 [mu] l of supernatant samples were used for the milk-clotting test.

It was further determined whether the milk-clotting activity of the sample is inhibitable by peps Latin or PMSF. The results are shown in Table 2.

Table 2

Results of the milk-clotting test + peps latin + PMSF

pJDBA / GRF18 - pJDB «.CH / GRF18. - - pJDBaCHE / GRF18 + - 4- chymosin (Serva) + - +

Example 4

Cloning of a synthetic gene or cDNA with synthetic additional sequences in pJDB and production of a protein secreted into the medium

An example of ensuring chymosin secretion using the pJDB vector, see Example 3, is one of the easiest cases to insert a foreign gene behind the MF signal sequence, as it is sufficient to place the prochymosin gene in the correct transition phase. The removal of the prochymosin prosequence here takes place autocatalytically (at medium pH = 5), independently of the protease activities of the S. cerevisiae secretory system. However, this possibility is only offered for some polypeptides and the procedure described (see Examples 2 and 3) cannot therefore be applied in general to ensuring the secretion of any foreign protein.

In this chapter, we describe, on the example of ensuring the secretion of human epidermal growth factor (hEGF), a method of construction enabling the secretion of the product of any synthetic preparation.

The construction requires that synthetically a gene starting with a codon for the first amino acid of the functional product and ending with a termination codon be set by short sequences on both sides. Figure 4 shows generally useful additional sequences that ensure easy insertion of synthetic DNA into the correct translation phase after the MFU signal sequence by substitution with the Hind HI-SalI fragment of the pJDB vector. In addition, the introductory additional sequence contains, immediately before the first codon of the foreign gene, a sequence determining Lys-Arg or Arg-Arg - the target site for the high-performance S. cerevisiae protease system encoded by the KEX2 gene. This protease ensures efficient secretion of the functional protein into the medium.

Giant. 4 i Example of an additional sequence ensuring the insertion of the synthetic hEGF gene into the pJDBct vector and the secretion of the functional product into the medium

KEX2

Ala Trp Light Arg ' Asn Ser and GGT TGG AAA AGA AAT Tea 'AjCC TTT TCT TTA AGG

HindIII additional sequence I.

encoding the hEGF gene sequence ·

Arg Term . CTG TAA © . GAG ATT jCAGCT

Hall

II.

Foreign genes of known sequence can be ligated in a similar manner. Using a suitable restriction endonuclease, the shortest possible introductory part of the gene is separated and replaced by a synthetic extension containing the additional sequence I, see Fig. 4, and the missing part of the gene. At the other end, a short adapter is connected, ending with a SalI site, similar to the additional sequence II, see Fig. 4, Note:

In some cases it is advantageous to use a _{HindIII-HindIII fragment of MFα instead of the additional sequence II t} see Fig. 4, the sequence containing HindIII, see Fig. 1. This method of foreign gene insertion allows to distinguish recombinant DNA-transformed clones from transformed with the recycled vector, since only these clones produce too much Λ-factor, are therefore detectable as described in Example 1.

Claims (6)

OBJECT OF THE INVENTION 1. A method for the secretory production of foreign proteins by culturing transformed Saccharomyces cerevisiae yeast cells, characterized in that the yeast cell is transformed with derivatives of the zJDBft zwitterionic vector carrying a combination of coding sequences with a yeast d-factor gene - MF », the combination of sequences being as follows - sequences ensuring the replication of the vector in Escherichia eoli, - sequences ensuring the replication of the vector in the yeast S. cerevisiae, - a sequence ensuring the selection of transformed E. eoli clones based on amp ^r a - sequences ensuring the selection of transformed S. cerevisiae leu2 ~ clones based on the complementation of the leu2 mutation, and the transformed cells are cultured and secrete functional chymosin into the medium. lo CS 27o7o3 Bl 2. The secretory production method according to item 1, characterized in that the derivatives of the vector pJDBa. contain a foreign coding sequence inserted into the MFα gene in the pJDBα vector, 3. The secretory production method according to items 1 and 2, characterized in that the derivatives of the pJDBa vector. labeled pJDBdCH and pJDBaCHE contain the calf prochymosin gene sequence appended to the MF 'signal sequence. in the pJDBa vector. 4. The secretory production method according to items 1 to 3, characterized in that the derivative of the vector pJDBa. labeled pJDBaCH contains a prochymosin sequence fused in the correct translational phase behind the MFα signal sequence and providing for the expression and secretion of functional chymosin in a S. cerevisiae yeast cell. ' 5. The secretory production method according to items 1 to 4, characterized in that the S. cerevisiae yeast cells transformed with pJDBttCHE produce and secrete functional chymosin into the medium. 6. The secretory production method according to item 1, characterized in that the foreign coding sequences are inserted into the pJDBa vector after the sequence encoding the amino acids recognized by the KEX2 protease system of S. cerevisiae.