MXPA01000516A - Method of making proteins in transformed yeast cells - Google Patents

Abstract

The present invention relates to a method for expressing heterologous proteins or polypeptides in yeast by culturing a transformed yeast strain which does not contain a functional antibiotic resistance marker gene.

Description

METHOD FOR THE PREPARATION OF PROTEINS IN TRANSFORMED YEAST CELLS FIELD OF THE INVENTION The present invention relates to the expression of proteins in transformed yeast cells, to the construction of DNA and to the vectors for use in such a process, and to the yeast cells transformed with the vectors.

BACKGROUND OF THE INVENTION It is well known to use the transformed yeast strains for the expression of proteins, see for example European patent applications Nos. 0088632A, 0116201A, 0123294A, 0Í23544A, 0163529A, 0123289A, 0100561A, 0189998A and 0195986A, the PCT patent applications Nos. WO 95/01421, 95/02059 and WO 90/10075, and U.S. Patent No. 4,546,082. It is a common feature of the above methods that the yeast production plasmid contains a gene for an antibiotic marker. Such marker gene is coming from the steps of Ref: 126130 initial cloning in E. coli where it was used to select the transformed cells or to maintain the plasmids used as vectors. Antibiotic marker genes are not believed to have any adverse impact on the culture of the transformed yeast cells and it has therefore been common practice not to take any steps to suppress such DNA. In addition, the characterization of the plasmidic construct is usually carried out by isolating the plasmids from the transformed yeast cells and transforming the isolated plasmid into E. coli followed by the selection with antibiotic. It has thus been convenient for practical purposes to retain the marker gene of antibiotic resistance. Although research laboratories and industrial production plants are controlled by very strict safety regulations there is always a small risk that a few cells per accident will be released into the environment. Due to its highly sophisticated nature, such genetically engineered microorganisms will only survive for a very short period and the risk of damaging the environment is extremely low. This is, of course, the reason why such transformed microorganisms have been approved for use in research and large-scale operations. Even if the cells die rapidly, the plasmids containing the antibiotic resistance gene can still be accidentally discarded into the environment and there is a theoretical risk of producing antibiotic resistance in bacteria if the plasmid is picked up spontaneously. Antibiotics are of great importance for the treatment of bacterial infections in humans and animals. Any risk of potential environmental contamination with a gene that confers resistance to antibiotics should be minimized, if possible. There is therefore a need to develop methods even safer than the methods used to date, and it is an object of the present invention to provide such improved methods.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a method for expressing heterologous proteins or polypeptides in yeast, wherein the yeast transforming strain used for production contains an expression vector in which an antibiotic marker gene used in the initial cloning steps has been made nonfunctional by in vitro modification before transformation of the yeast host. The present invention also relates to DNA sequences and expression vectors for use in such a method and to transformed yeast cells. According to one aspect, the present invention relates to a recombinant yeast expression vector, which is incapable of conferring antibiotic resistance to bacterial cells and which comprises a gene encoding a heterologous gene and a resistance marker gene. to antibiotics. , which has been made non-functional by in vitro modification. According to a further aspect, the present invention relates to a method for making a desired polypeptide or protein, said method comprises culturing a yeast strain comprising a vector that is incapable of conferring antibiotic resistance to the bacterial cells, and comprising a gene encoding a heterologous gene and an antibiotic resistance marker gene, whose marker gene has been non-functional by in vitro modification prior to transformation of the host yeast, and isolating the desired product from the culture medium. The method according to the invention will typically comprise the cultivation of a yeast strain containing an expression plasmid in yeast in whose plasmid a functional antibiotic marker gene, used for the initial cloning steps in bacteria, has been made non-functional by in vitro suppression of part of the marker gene or the complete marker gene prior to insertion into a host yeast to be used for the expression and secretion of the desired polypeptide or protein. The deletion of the antibiotic marker gene is preferably carried out by insertion of the appropriate restriction cleavage sites on each side of the antibiotic resistance marker gene, after which the marker gene is deleted by in vitro treatment with the enzymes of adequate restriction. The present invention is also related to transformed yeast strains comprising a vector that is incapable of conferring antibiotic resistance to bacterial cells, and which comprises a gene encoding a heterologous gene and an antibiotic resistance marker gene which it has been made non-functional by in vitro modification before the transformation of the yeast host. The yeast strain is preferably a strain of Sa ccharomyces, and in particular a strain of Saccharomyces cerevi siae. As used herein, the term "antibiotic marker gene" or "antibiotic resistance marker gene" means a gene that allows phenotypic selection of transformed bacterial cells and plasmid amplification. The antibiotic resistance marker genes most commonly used in E. coli are the marker genes that confer resistance against ampicillin (AMP), chloramphenicol, neomycin, kanamycin, and tetracycline.

As used herein, the term "non-functional marker gene" means that the marker gene has been either deleted or rendered non-functional by deletion of part of the gene. It is preferred that the gene be completely deleted. As used herein, "in vitro modification" means the modification steps used on the vector outside of the cell environment. As used herein, "unable to confer antibiotic resistance to bacterial cells" means that antibiotic resistance genes are non-functional in any organism due to the described genetic manipulation of the gene. As used herein, "yeast host" or "yeast host" means a yeast organism that is to be transformed or transfected with the plasmid or expression vector.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is further illustrated with reference to the accompanying drawings, wherein: Figure 1 shows the expression plasmid pAK729 which contains a gene expressing an insulin precursor under the control of expression of a TPI promoter and a TPI terminator sequence of S. cerevisiae and a signal leader sequence consisting of the signal peptide YAP3 and a synthetic peptide LA19. The construction of pAK729 is described in WO 97/22706. The plasmid also contains the AMP-R sequence of pBR322 / pUC13 which includes the ampicillin resistance gene and an origin of DNA replication in E. coli; Figure 2 shows the plasmid map of plasmid pAK729.5 used for the generation of strain NN729.5 lacking the AMP gene before the suppression of the Amp gene; Figure 3 shows the plasmid map of plasmid pAK729.6 used for the generation of strain NN729.6 that lacks the AMP gene before the suppression of the Amp gene; Figure 4 shows the plasmid map of the plasmid pAK729.6-? Amp in which the AMP gene has been deleted; Figure 5 shows the plasmid map of plasmid pAK729.7 used for the generation of strain NN729.7 that lacks the AMP gene before the suppression of the Amp gene; and Figure 6 shows the plasmid map of the plasmid pKV228 modified by the replacement of the coding sequence of EcoRI (940) - Xbal (1403) in pAK729 (Figure 1) with a coding sequence of MFalfa * -Arg3 GLP-I (7-37) • DETAILED DESCRIPTION OF THE INVENTION In vitro suppression of the antibiotic resistance marker gene is accomplished either by the use of suitable restriction sites or by introducing suitable restriction sites by the use of PCR, site-specific mutagenesis or other well-known techniques for manipulation of the DNA sequences, followed by treatment with the appropriate restriction enzymes. Four modified NN729 strains were constructed to assess whether several deletions in the plasmid can influence the fermentation performance of the insulin precursor or the stability of the strain during long-term fermentation (Table I). The strains were compared with the original strain NN729 with respect to the fermentation yield and the stability of the fermentation (Table II). In addition three strains of modified yeast were constructed that produce a variant of GLP-1, Arg3 GLP-l (7-37) constructed to evaluate if several deletions in the pKV228 plasmid containing the AMP gene can influence the fermentation performance of Arg34GLP-I (7-37) (Table III). The plasmids and strains where the AMP gene and possibly the neighboring sequences have been deleted, are all denoted "? AMP". The modified yeast strains were prepared by transforming the modified plasmids pAK729 or pKV228 in which the AMP marker gene and the other possible DNA sequences from the original plasmid had been deleted within the S strain. cerevi yes ae MT663 (E2-7B XE11-36 a / a,? tpi? tpi, pep 4-3 / pep 4-3) or ME1719 (MATa / a? yap3:: ura3 /? ayap3:: URA3pep4-3 / pep4-3? tpi:: LEU2 /? tpi:: LEU2 eu2 / leu2? ura / 3 /? ura3). The modified plasmids were prepared by using the appropriate restriction enzyme sites already present in the plasmid or by inserting suitable restriction enzyme sites in such a way that the AMP gene can be deleted. The modified plasmids can be manipulated in vitro before transformation into S. cerevi si a e (strain MT663) in such a way that the gene is suppressed or rendered nonfunctional and consequently the resulting yeast strain lacks the AMP gene. In this way, the potential risk for environmental contamination with the AMP gene during the disposal of the yeast cells is eliminated. Modified pAK729 or pKV228 plasmids were digested with the appropriate restriction enzymes, subject to agarose electrophoresis, isolated, re-ligated and subsequently transformed into the competent S. cerevisiae MT663 and ME1719 competent cells of WO98 / 01535, respectively. The protein or polypeptide produced by the method of the invention can be any heterologous protein or polypeptide that can be advantageously produced in a yeast cell. Examples of such proteins are aprotinin, the inhibitor of the tissue factor or other protease inhibitors, insulin, insulin precursors or insulin analogues, insulin-like growth factor I or II, human growth hormone or bovine, interleukin, tissue plasminogen activator, transforming growth factor aob, glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2), GRPP, Factor VII, Factor VIII, Factor XIII, growth factor derived from platelets, and enzymes, such as lipases. By "insulin precursor" or "a precursor of an insulin analog" is meant a single chain polypeptide, including proinsulin, which by one or more subsequent chemical and / or enzymatic processes can be converted to a double-chain insulin or an analogous insulin molecule that has the correct establishment of the three disulfide bridges as found in natural human insulin.The insulin precursors will typically contain a modified C-peptide that binds the A and B chains of insulin. of preferred insulin will lack the amino acid residue B (30) .The most preferred insulin precursors are those described for example in European patent EP 163529 and in PCT patent applications Nos. 95/00550 and 95/07931. of insulin are human insulin, preferably human insulin des (B30), and porcine insulin.The preferred insulin analogues are those in One or more natural amino acid residues, preferably one, two or three have been replaced by another codifiable amino acid residue. Thus, at position A21 a progenitor insulin can instead of Asn have an amino acid residue selected from the group comprising Ala, Gln, Glu, Gly, His, Lie, Leu, Met, Ser, Thr, Trp, Tyr, or Val, in particular an amino acid residue selected from the group comprising Gly, Ala, Ser, and Thr. Similarly, at position B28 a progenitor insulin can instead of Pro have an amino acid residue selected from the group comprising Asp, Lys, etc., and at position B29 a progenitor insulin can instead of Lys have the amino acid Pro . The term "a codifiable amino acid residue" as used herein, designates an amino acid residue that can be encoded by the genetic code, eg, a triplet ("codon") of nucleotides.

The DNA constructs used can be prepared synthetically by established standard methods, for example, the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3, 1984, pp, 801-805. According to the phosphoamidite method, the oligonucleotides are synthesized, in an automatic DNA synthesizer, purified, duplicated and ligated to form the synthetic DNA construct. A currently preferred way to prepare a DNA construct is by polymerase chain reaction (PCR), for example as described in Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, NY, 1989). The DNA encoding the desired protein may also be of genomic or cDNA origin, for example obtained by the preparation of a genomic or cDNA library and selecting the DNA sequences that code for all or part of the polypeptide of the invention by hybridization using synthetic oligonucleotide probes according to standard techniques (see Sambrook et al., Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor, 1989). Finally, the DNA coding for the desired protein can be of mixed synthetic and genomic origin, of synthetic origin and of mixed cDNA or of genomic origin and of mixed cDNA, prepared by annealing the fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the complete DNA construction, according to standard techniques. The recombinant expression vector can be a self-replicating vector, for example, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and is replicated together with the chromosome (s) into which it has been integrated. The expression system can be a simple vector or a plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The recombinant expression vector may contain a DNA sequence encoding the desired protein or the desired polypeptide, operably linked to a suitable promoter sequence. The promoter can be any DNA sequence that exhibits transcriptional activity in yeast and can be derived from genes encoding proteins either homologous or heterologous to the yeast. The promoter is preferably derived from a gene that codes for a protein homologous to yeast. Examples of suitable promoters are the Mal, TPI, ADH or PGK promoters of Saccharomyces cerevi si ae. The DNA sequence coding for the desired protein or for the desired polypeptide may also be operably linked to a suitable terminator, for example, the TPI terminator (see T. Alber and G. Kawasaki, J. Mol. Appl. Genet. , 1982, pp. 419-434). The recombinant expression vector of the invention will also comprise a DNA sequence that makes it possible for the vector to replicate in yeast. Examples of such sequences are the REP 1-3 replication genes of the 2μ yeast plasmid and the origin of replication. The vector may also comprise a selectable marker, for example, the TPI gene of Schi zosaccharomyces pombe as described by P.R. Russell, Gene 40, 1985, pp. 823-974. 125-130. Finally, the expression vector will preferably contain a signal / guide sequence to ensure the secretion of the desired protein or polypeptide into the culture medium. A signal sequence is a DNA sequence that codes for a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. The secretory signal sequence can code for any signal peptide that ensures the efficient direction of the expressed polypeptide towards the secretory pathway of the cell. The signal peptide may be the signal peptide of natural origin, or a functional part thereof, or it may be a synthetic peptide. Signal peptides useful for yeast host cells are obtained from the genes for the a factor of Saccharomyces cerevi si ae and the invertase of Saccharomyces cerevi si ae, the signal peptide of salivary amylase of mouse (see 0. Hagenbuchle et al., Nature 289, 1981, pp, 643-646), a modified carboxypeptidase signal peptide (see LA Valls et al., Cell 48, 1987, pp. 887-897), the BARÍ yeast signal peptide ( see WO 87/02670), or the signal peptide of yeast aspartic protease 3 (YAP3) (see M. Engel-Mitani et al., Yeast 6, 1990, pp. 127-137). For efficient secretion in yeast, a sequence encoding a leader peptide can also be inserted downstream (3 ') of the signal sequence and upstream (5') of the DNA sequence encoding the polypeptide. The function of the guiding peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (eg export of the polypeptide through the cell wall or at least through the cell membrane towards the periplasmic space of the yeast cell). The guiding peptide can be the guide for yeast a-factor (the use of which is described for example in US Patents 4,546,082, EP 16,201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic guide peptide which is a guiding peptide not found in nature. Synthetic guiding peptides can be constructed as described in WO 89/02463 or WO 92/11378 and by Kjeldsen et al in "Protein Expression and Purification 9, 331-336 (1997). L expression" leader or guide peptide " it is understood that it indicates a peptide in the form of a propeptide sequence whose function is to allow the heterologous protein to be secreted to be directed from the endoplasmic reticulum to the Golgi apparatus and also to a secretory vesicle for secretion into the medium, ( example export of the expressed protein or the expressed polypeptide through the cell membrane and the cell wall, if present, or at least through the cell membrane into the periplasmic space of a cell having a cell wall).

The methods used to ligate the DNA sequences encoding the desired protein or for the desired polypeptide, promoter and terminator, respectively, and to insert them into suitable yeast vectors containing the information necessary for yeast replication , are well known to those skilled in the art (see for example, Sambrook et al. op. cit.). It will be understood that the vector can be constructed either by first preparing a DNA construct containing the complete DNA sequence encoding the polypeptide of the invention, and subsequently by inserting this fragment into a suitable expression vector, or by the sequential insertion of the DNA fragments that contain the genetic information for the individual elements (such as the signal, guiding or heterologous protein) followed by the ligation. The yeast organism used in the process of the invention can be any suitable yeast organism, which, after culturing, produces satisfactory amounts of the desired protein or polypeptide. Examples of suitable yeast organisms may be strains selected from the yeast species Saccharomyces cerevi siae, Saccharomyces kl uyveri, Schi zosaccharomyces pombe, Saccharomyces uvarum, Kl uyveromyces l acti s, Hansenula poly orpha, Pi chia pastoris f Pichia methanolica, Pichia kl uyveri, Yarrowia lipolyti ca, Candida sp. , Candi da utili s, Candida cacao! , Geotri chum sp. , and Geotri chum fermen tans, preferably the yeast species Saccharomyces cerevi siae. The transformation of the yeast cells can be effected, for example, by protoplast formation, followed by transformation in a manner known per se. The medium used to grow cells can be any conventional medium, suitable for the development of yeast organisms. The secreted heterologous protein, a significant proportion of which will be present in the medium in properly processed form, can be recovered from the medium by conventional methods including the separation of the yeast cells from the medium, by centrifugation or filtration, precipitating the components Proteins of the supernatant or filtrate by means of a salt, for example, ammonium sulfate, followed by purification by a variety of chromatographic procedures, for example, ion exchange chromatography, affinity chromatography, or the like. When the protein is secreted into the periplasmic space, the cells are enzymatically or mechanically disintegrated. The desired protein or polypeptide can be expressed and secreted as an extended fusion protein at the N-terminus as described in WO 97/22706. The N-terminal extension can then be removed from the protein recovered in vitro, by chemical or enzymatic cleavage, as is well known in the art. It is preferred to drive cleavage by the use of an enzyme. Examples of such enzymes are trypsin or proteolyase I of Achromobacter lyti cus. The present invention is described in further detail in the following examples which are not intended to be in any way limiting the invention as claimed.

Example 1 The yeast plasmid pAK729 constructed for the expression of an insulin precursor (an insulin precursor B (1-29) -Ala-Ala-Lys-A (1-21) extended at the N-terminus, see WO 97/22706) It contains two restriction sites for ApaLI, Apall (4477) and Apall (5723) (see Figure 1). These restriction sites are located on either side of the AMP marker gene. Removal of the 1246 nucleotides between the ApaLI sites in pAK729 will remove the AMP marker gene and some of the extra plasmid DNA derived from E. coli. Plasmid pAK729 was digested with ApaLI restriction enzyme, subject to agarose electrophoresis, isolated, religated and subsequently transformed into competent S cells. cerevi si ae (MT663, see European patent EP B0163529) to give the transformed yeast strain NN729.1-? AMP. The modified expression plasmid was reisolated from the yeast strain NN729.1ΔAMP and the DNA sequences were verified after generation by PCR followed by subcloning of the DNA region characterizing the deletion. Similarly, the DNA sequences encoding the insulin precursor were checked on the reisolated plasmid DNA of the yeast strain NN729.1? AMP. Yeast strain NN729.1? AMP was cultured in YPD medium at 30 ° C for 72 hours. The fermentation yield of the insulin precursor was determined by reverse phase high resolution liquid chromatography (RP-CLAP).

Example 2 The enzyme restriction sites, Xhol (5676) and Xhol (5720) were introduced into plasmid pAK729 by PCR. The DNA sequences selected from the resulting plasmid pAK729.5 were subsequently verified. The restriction plasmid map of pAK729.5 is shown in Figure 2. The DNA fragment between the restriction enzyme sites, Xhol (5676) and Xhol (5720) can be deleted by the plasmid pAK729.5 by deleting 44 nucleotides located within the AMP gene. Plasmid pAK729.5 was digested with Xhol restriction enzymes, subject to agarose electrophoresis, isolated, religated and subsequently transformed into competent S cells. cerevi si a e MT663 giving the yeast transformant NN729.5-? AMP. The modified expression plasmid was reisolated from the yeast strain NN729.5-? AMP and the DNA sequences were verified after generation by PCR, followed by subcloning of the DNA region characterizing the deletion. Similarly, the DNA sequences encoding the insulin precursor were checked on the reisolated plasmid DNA of the yeast strain NN729.5-? AMP. Deletion of nucleotide 44 in pAK729.5-? AMP appeared to be as efficient as a complete deletion of the AMP gene with respect to the destruction of β-lactamase activity. Yeast strain NN729.5-? AMP was cultured in the YPD medium at 30 ° C for 72 hours. The fermentation yield of the insulin precursor was determined by RP-CLAP.

Example 3 The restriction enzyme site, AatlI (4982), was introduced into plasmid pAK729 by PCR. The DNA sequences selected from the resulting plasmid pAK729.6 were subsequently verified. The restriction plasmid map of pAK729.6 is shown in Figure 3. In pAK729.6 the DNA fragment between the restriction enzyme sites AatlI (4982) and AatlI (5978) can be deleted, removing 996 nucleotides from the plasmid . This will eliminate the entire AMP gene and the promoter. Plasmid pAK729.6 was digested with the DNA restriction enzyme AatlI, subject to agarose electrophoresis, isolated, religated and subsequently transformed into the competent S cells. cerevi yes ae MT663. The modified expression plasmid was reisolated from the yeast strain NN729.6? AMP and the DNA sequences were verified after generation by PCR, followed by subcloning of the DNA region characterizing the deletion. Similarly, the DNA sequences encoding the insulin precursor were checked on the reisolated plasmid DNA of the yeast strain NN729.6-? AMP. Plasmid pAK729.6-? AMP lacking the AMP gene is shown in Figure 4. Yeast strain NN729.6-? AMP was cultured in YPD medium at 30 ° C for 72 hours. The fermentation yield of the insulin precursor was determined by RP-CLAP.

Example 4 The new restriction enzyme site, AatlI (3801), in plasmid pAK729.7 was introduced into the original plasmid pAK729 by PCR. The DNA sequences selected from plasmid pAK729.7 were subsequently verified. In pAK729.7 the DNA fragment between the restriction enzyme sites AatlI (3801) and AatlI (5978) can be deleted, removing 2177 nucleotides from the expression plasmid. Plasmid pAK729.7 was designed so that the AMP gene and the origin of replication of E. col i can be deleted. The restriction plasmid map of pAK729.7 is shown in Figure 5. Plasmid pAK729.7 was digested with the DNA restriction enzyme AatlI, subject to agarose electrophoresis, isolated, religated and subsequently transformed into competent cells of S. cerevi si a e MT663. The modified expression plasmid reisolated from the yeast strain NN'729.7-? AMP and the DNA sequences were verified after generation by PCR, followed by subcloning of the DNA region characterizing the deletion. Similarly, the DNA sequences encoding the insulin precursor were checked on the reisolated plasmid DNA of the yeast strain NN729.7-? AMP. Yeast strain NN729.7-? AMP was cultured in YPD medium at 30 ° C for 72 hours. The fermentation yield of the insulin precursor was determined by RP-CLAP.

Table I Description of NN729 strains based on plasmids pAK729 with non-functional or deleted AMP gene The new NN729-? AMP strains were compared with the original strain NN729 with respect to the fermentation yield of the insulin precursor (Table II).

Table II Fermentation performance of NN729-? AMP strains transformed with plasmids with non-functional or deleted AMP gene From the above it appears that yeast strains comprising an expression plasmid with a partially or completely deleted AMP marker gene, express 10-20% more of the insulin precursor compared to the original yeast strain comprising an expression plasmid which contains the AMP gene.

Example 5 Expression of Arg34GLP-l-7-37) in yeast using the plasmids with the non-functional or deleted AMP resistance gene The sequence EcoRI (940) -Xbal (1403) of the pAK729 constructs coding for LA19 X5 M13 illustrated in Figures 1-5 were replaced with a coding sequence MFalfa * -Arg34GLP-l (7-37) for the present example (Figure 6). A modification of the pre-pro guide peptide MFal (Kurjan &Herskowitz, Cell 30, 1982, pp. 933.) in which the Leu at position 82 and Asp at position 83 have been replaced with Met and Ala respectively, introducing the Ncol cleavage site in the DNA sequence was applied in this construction. The guide sequence was designated MFal * (Kjeldsen T. et al., 1996). The MFal * signal peptide sequence of the MFal signal includes the dibasic recognition portion Kex2p (Lys-Arg) which separates the guide from the coding sequence for Arg34GLP-I (7-37). The peptide Arg34GLP-l (7-37 j is a variant of GLP-1 (7-37 | human (S. Mojsov, et al., J Biol Chem. 261, 1986, pp. 11880-11889) wherein the residue of natural amino acid at position 34 is substituted with an Arg residue. Three Arg34GLP-I (7-37) expression plasmids were constructed with disintegrations of the AMP resistance gene as described for NN729.1 (Example 1), NN729. 5 (Example 2) and NN729.6 (Example 3) and subsequently transformed into S. cerevi if ae competent cells ME1719 (see WO 98/01535) giving the yeast transformants YES2076, YES2079 and YES2085, respectively. , which has been used to express Arg3 GLP-l (7-37), is a diploid strain and has phenotypes that lack two aspartyl proteases, for example (1) yeast aspartyl protease 3 (YAP3) which breaks the C-terminal side of the mono- or dibasic amino acid residues (Egel-Mitani; and collaborators YEST 6: 127-137, 1990) and (2) l a vacuolar protease A responsible for the activation of other proteases such as protease B, carboxypeptidase Y, aminopeptidase I, RNAse, alkaline phosphatase, acid trehalase and exopolyphosphatase. In addition, the triose phosphate isomerase (TPI) gene has been disintegrated, whose phenotype makes it possible to use glucose in transformants developed on glucose-containing medium. The genetic background ME1719 is MATa / a Dyap3:: ura3 / Dyap3:: URA3 pep4-3 / pep4-3 tpi:: LEU2 / Dtpi:: LEU2 Ieu2 / leu2 Dura3 / Dura3. The modified expression plasmids pKV301, pKV307, and pKV304 were reisolated from yeast strains, and the DNA sequences were verified after generation by PCR, followed by subcloning of the DNA region characterizing the suppression. Similarly, the DNA sequences encoding Arg34GLP-I (7-37) were verified on the reisolated plasmid DNA of the yeast strains. Table III shows a comparison between the modified and unmodified strains.

Table III Description of YES strains based on plasmids with non-functional AMP resistance gene or suppressed for the expression of Arg34GLP-I (7-37) The yields were compared from fermentation from fermentation at a laboratory scale of 5 ml, in YPD for 72 hours at 30 ° C. The yields were evaluated using CLAP.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (8)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A recombinant yeast expression vector that is incapable of conferring antibiotic resistance to bacterial cells, the vector is characterized in that it comprises a gene encoding a heterologous gene and an antibiotic resistance marker gene which has been rendered non-functional by modification in vitro

2. The expression vector in recombinant yeast according to claim 1, characterized in that the antibiotic marker gene is the AMP gene.

3. A method for making a desired polypeptide or protein, characterized in that it comprises culturing a yeast strain comprising a vector according to claims 1-2 under suitable conditions and isolating the desired product from the culture medium.

4. A method for making a desired polypeptide or protein, characterized in that it comprises • the cultivation under suitable conditions of a yeast strain containing a yeast expression plasmid in whose plasmid a functional antibiotic marker gene, used for the initial cloning steps in bacteria, has been rendered non-functional by in vitro suppression of part of the marker gene or the complete marker gene prior to insertion into the yeast host.

5. The method according to claim 4, characterized in that it comprises inserting suitable restriction sites around the antibiotic resistance marker gene and suppressing the marker gene by in vitro treatment with suitable restriction enzymes.

6. The method according to claims 3-5, characterized in that the yeast strain is a strain of Sa ccharomyces, preferably a strain of Sa ccharomyces cerevi si ae.

7. The transformed yeast cell, characterized in that it comprises a vector according to claims 1-2.

8. The transformed yeast cell according to claim 7, characterized in that it is a strain of Sa ccharomyces, preferably a strain of Saccharomyces cerevi siae.