EXPRESSION VECTOR FOR IMPROVED PRODUCTION OF POLYPEPTIDES IN YEAST

Abstract: A new expression vector for the production of a polypeptide in yeast comprising a sequence coding for said polypeptide, and other sequences allowing expression of said polypeptide only in yeast, said other sequences lacking any non-yeast sequences, a yeast strain comprising such a vector, a method for the production of such new expression vector, a method for the production of a yeast strain comprising transformation of a yeast strain with such new vector and a method for the production of a polypeptide in such transformed yeast strain comprising fermentation thereof and isolating the polypeptide.

Description: EXPRESSION VECTOR FOR IMPROVED PRODUCTION OF POLYPEPTIDES IN YEAST Field of the Invention The invention concerns a new expression vector for the production of a polypeptide in yeast, a yeast strain being transformed with such vector, methods for the production of said vector, said yeast strain and said polypeptide. BACKGROUND OF THE INVENTION In genetic engineering techniques for expression in yeasts are commonly used shuttle vectors comprising a nucleotide sequence coding for a particular polypeptide in combination with sequences necessary for said expression in yeast, such as a yeast promoter. Such shuttle vectors comprise also sequences allowing expression in bacteria, such as usually Escherichia Coli, or other microorganisms. Such additional non-yeast sequences are only useful for the construction of the vectors. However, they are superfluous for the expression in yeast. In fact they may hinder the efficient expression of the polypeptide in yeast or retard the replication of the organism because the superfluous nucleotides must also be doubled, which is an energy consuming process. The yeast Saccharomyces Cerevisiae is usually used as an excellent microorganism for the production of both homologous and heterologous proteins because of its well-known genetic system, rapid growth and technical advantages of manipulation. In addition, the development of DNA transformation systems for the introduction of cloned genes and their inexpensive and safe overproduction in simple fermentation conditions, has made this organism particularly useful for large-scale industrial practice. A number of yeast polypeptides are known in the art. Of particular interest are the superoxide dismutases. The yeast Saccharomyces cerevisiae contains two species of superoxide dismutases (EC 1.15.1 1), the copper/zinc-(Cu/Zn SOD) and the manganese-(Mn SOD) containing forms. The former one (Cu/Zn SOD) is localized in the cytoplasm while the manganese enzyme is restricted to the mitochondrial matrix. This enzyme is assumed to provide protection in vivo against free toxic radicals generated within cells as intermediates of normal metabolism (Bilinski, T. et al. Biochem. Biophys. Res. Commun. 130: 533-539 (1985), Van Loon A.P.G.M. et al. Proc. Natl. Acad. Sci. USA 83: 3820-3824 (1986), Lee F.J. et al. J. Free Rad. Biol. Med. 1 : 319-325 (1985), Galiazzo F. et al. Biochim. Biophys. Acta 965: 46-51 (1988)). Consequently, it is expected to be used in preventing or treating potential damages in human, in particular damages from cell aging and senescence (Rosen D.R. et al. Nature 362, 59-62 (1993), McCord J. M. and Fridovich I. J. Biochem 244: 6049-6055 (1969),. McCord J.M. et al. Proc. Natl. Acad. Sci.USA 68: 1024-1027 (1971), McCord J. M. N. Engl. J. Med. 312: 159-163 (1985)). The Cu/Zn SOD gene of the yeast Saccharomyces Cerevisiae has been cloned and sequenced (Bermingham-McDonogh O. et al. Proc. Natl. Acad. Sci. USA 85: 4789-4793 (1988)) and the structure and mechanism of action of the enzyme is well characterized (Djinovic K. et al. J. Mol. Biol. 225: 791-809 (1992), O'Neill P. et al. Biochem. J. 251 : 41- 46 (1988)). The Cu/Zn SOD is an abundant metalloenzyme present in the cytoplasm of most aerobic and many anaerobic organisms, whose activity catalyses the dismutation of the superoxide anion to dioxygen and hydrogen peroxide. OBJECT OF THE INVENTION It is an object of the present invention to improve on the yields of polypeptides in the fermentation processes of yeasts transformed with expression vectors coding for such polypeptides. It is a further object to provide such new vectors which are able to express the desired polypeptide yeast in larger amounts compared to the processes used before. A further object are new yeast strains which are transformed with such vectors and which are superior compared to the wild type or which are transformed with a shuttle vector. SUMMARY OF THE INVENTION According to the present invention an expression vector is provided, as well as a yeast strain, in particular a Saccharomyces Cerevisiae strain, transformed with said vector, which produces higher levels of a yeast or non-yeast polypeptide, e.g. an enzyme, such as e.g. Cu/Zn Superoxide Dismutase, than the wild type or the strain transformed with a shuttle vector. Methods for the preparation of such expression vector, yeast strains and endogenous yeast polypeptides are set forth. Numerous aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the detailed description and the drawings of the invention which provides illustrations of the practice of the invention in its embodiments. DETAILED DESCRIPTION In the present invention the following primer DNA sequences have been used the structures of which are precisely shown in the Sequence Identification Listing (the bp regions of the primers were taken from the EMBL vector, Gene Bank accession No JO3279): SEQ ID NO 1: is the external primer SOD-3, upstream region 81-102 bp SEQ ID NO 2: is the external primer SOD-2, downstream region 1018-1036 bp SEQ ID NO 3: is the internal primer SOD-4, region 971-991 bp Following is a short description of the drawings which serve to more easily comprehend the present invention: FIG. 1 shows an abstract of the coding region of the Cu/Zn SOD gene from yeast strain S288C with the positions of the external primers SOD3 and SOD2 and the internal primer SOD4 designed on the upstream and downstream regions of the SOD gene locus. FIG. 2 shows the new molecular plasmid construct pEMBL-SOD 374, which is plasmid pEMBLyex4 with a 374 bp upstream region, the coding sequence and a downstream region of the yeast Cu/Zn SOD gene under the control of the yeast GAL/CYC promoter. FIG. 3 shows a map of the final plasmid pEMBL-SOD w/o MCS w/o Coli comprising a 374 upstream region, the coding sequence and a downstream region of the yeast Cu/Zn SOD gene between the restriction sites EcoRI and Hindll, under the control of the yeast GAL/CYC promoter, it comprises only yeast sequences and replicates only in yeast. The present invention concerns in a first embodiment a new expression vector for the production of a polypeptide in yeast comprising a sequence coding for said polypeptide, and other sequences allowing expression of said polypeptide only in yeast, said other sequences lacking any non-yeast sequences. By the term expression vector is intended a vector, in particular a DNA vector, such as a plasmid which comprises a sequence coding for a polypeptide, a promoter sequence in reading frame with the coding sequence, and optionally other sequences, which are needed for efficiently producing or using the vector, such as an ori, a leader sequence, a terminator and a sequence coding for a polypeptide for selection of the vector. Such sequences are only derived from yeasts and are well known in the art. The sequence coding for said polypeptide is a yeast or a non-yeast sequence. Yeast sequences are for example such coding for a yeast polypeptide with enzyme functions, e. g. an antioxidative enzyme, such as superoxide dismutase SOD, thiol specific antioxidant TSA, or cytochrome c peroxidase, proteases, e. g. cerevisin precursor PRB1, proteinase inhibitors, e. g. proteinase B inhibitor 2, cytokines, and the like. Sequences coding for a non-yeast polypeptide are derived from any living organism, in particular from humans and animals. Such polypeptides are preferably useful in the medical art and comprise human insulin, tissue plas inogen activator, interferons, erythropoietin, growth factors, e.g. keratinocyte growth factor, enzymes, e. g. tryptase, Protein C activator, enzyme inhibitors, e. g. .tissue inhibitors of metalloproteinases (TIMP's), elastase inhibitors, and the like. The sequences coding for such useful polypeptides are known in the art. All these sequences are under the control of a yeast promoter. Useful yeast promotors are known in the art and comprise e. g. the GAL/CYC promoter. The final vector of the invention is only replicable in yeast cells. With the exception of the non-yeast sequence coding for any desired non-yeast polypeptide, it is lacking any non-yeast sequences. A preferred vector according to the invention is a yeast plasmid comprising the Cu/Zn SOD gene which is under the control of the GAL/CYC promoter, and is in particular the plasmid named pEMBL-SOD,w/oMCS,w/oColi. This plasmid can be used as a starting plasmid for constructing an expression vector where the Cu/Zn SOD gene is exchanged against a sequence coding for any other polypeptide. In a further embodiment the invention provides a method for the production of a new expression vector defined hereinbefore. The method for the production of a new expression vector according to the invention is characterized in that from a shuttle vector able to express a polypeptide in a yeast strain any non-yeast sequences are excised, and optionally the sequence coding for said polypeptide is replaced by a sequence coding for another polypeptide. The new expression vectors are obtained in a conventional manner from known shuttle vectors, such as the yeast integration plasmid YIp, the yeast replication plasmid YRp, the yeast centromeric plasmid YCp or the yeast episomeal plasmid YEp which are constructed to comprise a polypeptide gene and lack any non-yeast DNA sequences. The starting shuttle vectors may already comprise the sequence coding for the desired polypeptide under the control of any yeast promoter, e.g. the GAL/CYC promoter. Such vector is for example the plasmid pEMBL-SOD 374 or pEMBL-SOD ATG. These plasmids are used as intermediates for the production of the final vector according to the invention. If the gene is not yet available, the construction starts with the isolation of the gene coding for the desired polypeptide from a known source containing it, e. g. from a human or animal or a wild type microorganism strain. The gene is multiplied by the wellknown PCR method by means of synthetic primers, and inserted into the vector, usually a shuttle vector. From the intermediate vectors all non-yeast sequences are deleted, such as bacterial sequences, including multiple cloning sites, such as the bacterial origin of replication ORI, selectable markers, the origin of replication of the filamentous bacteriophage fl, the Ampicillin resistant gene and the like, making use of common restriction enzymes. Following is amore detailed discussion of the method used: Gene expression requires placing the gene, which is the DNA coding for the polypeptide of interest, under the control of a strong yeast promoter that will direct the synthesis of the corresponding messenger RNA. The DNA regulatory elements required for expression are carried by yeast vectors. These vectors are "shuttle vectors" that may be propagated in yeast strains as well as in the bacterium Escherichia coli for convenient manipulations and large scale preparations of the different intermediate plasmids. A number of different yeast integrating (YIp), replicating (YRp), centromere (YCp) and episomal (YEp) plasmid vectors have been developed ( Rose A.B., Broach J.R. Methods in Enzymology, 185:234-279 (1990), Schneider J.C., and Guarente L. Methods in Enzymology, 194: 373-388 (1991)). The plasmid that was chosen for the expression of the Cu/Zn SOD gene is the specific YEp (yeast episomal plasmid) shuttle vector pEMBLyex4 of 8.800 base pairs (Cesarani and Murray, in Setlow J.K. (ed) Genetic Engineering: Principle and Methods, Volume 9, Plenum Press, NY 134-135 (1987)). Such a vector carries the 2-micron yeast episome (a small double-stranded DNA plasmid present in the nuclei of most Saccharomyces Cerevisiae strains) which provides high mitotic stability and the ability to be autonomously replicated (Murray J.A.H., Mol. Microbiol., 1 : 1-4 (1987), Hartley and Donelson, Nature, 286: 860-864 (1980), Clark-Walker G.D. and Miklos G.L.G., Eur. J. Biochem., 41 : 359-365 (1974), Futcher A.B., and Cox B.S. J., Bacteriol., 157: 283-290 (1984)). The persistence of the plasmid is due to the presence, in the 2-micron moiety, of the REP 3 locus (for the partitioning of the plasmid during cell division) and the ARS sequence (origin of replication). The plasmid pEMBLyex4 carries the LEU 2 and URA 3 selectable markers which are extremely useful both to select the initial yeast cell transformants and to provide constant pressure to maintain the plasmid in the yeast cell (Alani E. et al., Genetics., 1 16: 541 (1987), Gritz L. and Davies J., Gene, 25: 179 (1983), Raster K.R. et al., Curr. Genet., 8: 353 (1984), Rine J. et al., Proc. Natl. Acad. Sci., USA, 80:6750 (1983)). In general, however, these kinds of plasmids achieve a good maintenance even in the absence of positive selection. In such a situation, cells can lose the plasmid at a rate of about 4 percent per generation. The pEMBLyex4 forms part of a special class of 2-micron vectors with a very high copy number (about 100-200 per cell). Moreover, yeast strains lacking the 2-micron episome (cir°) to propagate the plasmid were used. The stability of pEMBLyex4 in such strains is known to be very high even without continued selection pressure. The pEMBLyex4 plasmid includes the entire yeast expression hybrid cassette UAS GAL/CYC. The promoter cassette contains an upstream activation site (UAS sequence) and the promoter region (TATA box) for both high level of transcription of the downstream gene and regulation of the expression, a multiple cloning site (MCS) for inserting the gene and a termination region (Guarente L. et al., Proc. Natl. Acad. Sci, USA, 79: 7410-7414 (1982)).The hybrid cassette UAS gal/CYC carries from 5' to 3' the following regions: a 365 bp fragment (Sau3A-XhoI) from the upstream activation sequence of the region between the yeast GAL4 and GAL 10 genes which contains the binding region for the GAL4 product; a 250 bp region (XhoI-SstI) containing the promoter of the yeast gene CYC1, which carries the TATA box and the mRNA start sites but without the ATG region; a polylinker (Sstl-Hindlll) of 95 bp with unique restriction enzyme sites; a 250 bp region (in a Hindlll-SnaBI fragment) carrying polyadenylation and transcription terminator signals, from the 2-micron FLP gene. The expression system is regulated by the GAL4 and GAL80 gene products. GAL4 protein is a transcriptional activator that binds to the UAS gal sequences. The activity of the GAL 4 protein is inhibited by the binding of the GAL80 protein to its carboxy terminal region. The system is repressed by glucose, which inhibits the binding of GAL4 protein to the UASgal, and is induced by galactose, which causes the dissociation of the GAL80 protein from the GAL4 protein. In a further embodiment the invention provides a novel yeast strain transformed with an expression vector according to the invention Yeast species are any known species useful for the expression of yeast or non-yeast polypeptides, for example Saccharomyces cerevisiae or Saccharomyces occidentalis, or non-Sacheromyces yeast species, e. g., Hansenula polymorpha, Pichia pastoris, Schwanniomyces occidentalis, and Pichia stipitis. A preferred novel yeast strain is e.g. Saccharomyces cerevisiae, with improved ability to synthesize the Cu Zn SOD enzyme through insertion of the relative homologous gene in the intracellular compartment. Preferred intermediate yeast strains according to the present invention are for example GRF 18 transformed with plasmid pEMBL-SOD 374 or plasmid pEMBL-SOD ATG which have been produced, isolated and characterized. A further object of the invention is a method for the production of a yeast strain transformed with an expression vector coding for an endogenous yeast polypeptide, lacking any non-yeast sequences and being able to overproduce said yeast polypeptide in said yeast strain characterized in that a yeast strain is transformed with a new vector described hereinbefore. The transformation follows methods common in the art, such as the LiCl method of Ito et al., e. g. modified by R.H.Schiestl et al. (1989), Current Genetics. 16, 339-346, or the method of Hinnen et al.. (1978), Proc. Natl. Acad. Sci. USA, 75, 1929-1933. In a further embodiment the invention provides a method for the production of a polypeptide in a yeast strain comprising fermentation of a yeast strain transformed with an expression vector according to the invention. Fermentation follows methods common in the art, such as the fed-batch method with a controlled fed of glucose during the growth phase and induction of expression by addition of galactose in the middle of the growth phase according to Alberghina, Lilia, et al., (1991), Biotech, and Appl. Biochem. 14, 82 - 92. The following examples are presented by way of illustration of the invention and are directed to procedures carried out for the isolation and characterization of a yeast enzyme gene, e.g. Cu/Zn SOD gene, from traditional strains of S. Cerevisiae, to procedures for expression of the yeast enzyme gene in yeast strains under the control of a strong yeast promoter and to development and characterization of a yeast strain able to produce high levels of the enzyme,e.g. SOD protein. Abbreviations Hereinbefore and hereinafter the following abbreviations are used: ARS Autonomously Replicating Sequence EDTA ethylenediamine tetra acetic acid EMBL European Molecular Biology Laboratory LB Luria Bertani Medium MCS multiple cloning site PCR polymerase chain reaction PIU Pyrogallol Inhibitory Units REP Replikon (short DNA-sequence which serves in cells as origin of DNA replication) SDS sodium dodecyl sulfate SOD superoxide dismutase TBE Tris-Borate-EDTA buffer TE Tris-EDTA buffer w/oColi without Escherichia coli sequences w/oMCS without multiple cloning site YCp yeast centromeric plasmid YEp yeast episomal plasmid YEPD Yeast Extract-Peptone-Dextrose medium YIp yeast integration plasmid Yrp yeast replication plasmid Example 1: Extraction and purification of yeast genomic DNA This example relates to the extraction and purification of yeast genomic DNA to be used as genetic source for the isolation of the yeast Cu/Zn SOD gene. The DNA source to isolate the Cu/Zn SOD gene can be any yeast wild type strain. In this specific case DNA was extracted from Saccharomyces cerevisiae strains S288C, wild type, gal2, and W309, wild type. Similar DNA extraction could be performed by using other wild type yeast species, e.g. W303 as a DNA source. The haploid strain S288C is a typical strain of yeast which is currently used in most of the Molecular Biology laboratories around the world for the isolation of yeast genes and genetic and biochemical studies (Mortimer R.K. and Johnson J.R., Genetics, 113:13 (1986)). The strain W309 has been considered as a potential alternative source. Both strains are known to carry a copy of the wild type Cu/Zn SOD gene in their genome. The strains were provided by The Departement of Physiology and General Biochemistry, University of Milan. The protocol that was used to extract total yeast genomic DNA was performed according to modified methods of Cryer, Ecclesial and Marmur, Methods Cell Biology, 12:39-44 (1975) as following: Yeast cells, from a petri plate with YEPD medium, were inoculated in 200 ml of complete medium YEPD (1% Bacto-yeast extract, 2% Bacto-peptone, 2% Dextrose) and grown in a 1 liter flask, over night at 30° C, under shaking until late exponential phase (about 8x10' cells/ml). A total of 8x10 8 cells were used for the extraction of the DNA. 10 ml of cells were spinned out and concentrated in a polypropylene tube and the pellet was transfered to a 1.5 ml Eppendorf tube. 300 microliters of lysis buffer (NaCl 0.15 M, EDTA 0.1 M pH 8, SDS 1%) and 300 microliters of glass microbeads (diameter of 0.5 mm) were added to the Eppendorf tube. The cells were vortexed five times on ice, with pauses of 1 minute: The cell suspension was homogenized by vortexing with 600 microliters of phenol- chloroform-TE solution and spinned for 2 minutes. The upper aqueous phase was transfered to a new Eppendorf tube and 600 microliters of chloroform-isoamylic-alcohol solution (24:1) was added and mixed. The upper phase, transfered to a new tube, was incubated at 37 °C for 30 minutes with RNAase as a final concentration of 1 mg/ml. After ethanol precipitation the pellet was resuspended in T.E. buffer (Tris 10 mM, EDTA 1 mM, pH 8). About 100 mg of yeast genomic DNA with concentration of about 1 mg/ml were obtained from each preparation following this method, enough for many experiments. Example 2: Isolating the chromosomal region carrying the Cu/Zn SOD gene Polymerase Chain Reaction (PCR) was used to isolate the chromosomal region carrying the Cu Zn SOD gene. The Cu/Zn SOD gene maps the right arm of chromosome X in S. Cerevisiae between the cycl-rad6-SUP4-cdc8 cluster and cdcl l region (Chang et al. J. Biol. Chem. 266: 4417- 4424 (1991). It has no introns in its coding sequence and, consequently it is possible to isolate the entire translated region directly from genomic DNA by PCR. In addition, both the 5' upstream region and the 3' downstream region of the gene are known. To perform the PCR reaction, couples of synthetic oligonucleotides (primers) which span the SOD gene between the upstream and the downstream region are needed. The primers for the PCR were designed using the OLIGO primer analysis software, Version 4.0 (National Biosciences Inc. Plymouth) to cover the region of the published Cu/Zn SOD gene sequence of 1037 bases in Bermingham-McDonogh 0, Gralla E, Valentine J. PNAS. 85, p. 4789 (1988) (EMBL/Gene Bank, accession n° J03279). The primers were synthesized on an Applied Biosystem 392 Nucleic Acid synthesizer (Perkin-Elmer Corp., Foster City, CA) and purified by gel filtration with Sephadex G-25 DNA grade NAP -25 Columns (Pharmacia P-L Biochemicals Inc; Milwaukee, Wisconsin). As general strategy aimed to increase the chance to isolate the Cu/Zn-SOD gene from the yeast genome the so called "semi-nested PCR" was used. This strategy uses a two-step protocol which needs three different primers (FIG. 1). The first step, which uses two primers (one upstream and one downstream primer, e.g. SOD-3 and SOD-2), allows the amplification of a larger region of the genome. This step is followed by a second amplification, which uses a new internal primer (SOD-4) and one of the previous two (SOD-3), which finally permits the isolation and recovery of the SOD gene. The first round of PCR amplification was performed using the following primers: SOD-3 (upstream primer, region 81-102 of the sequence entered in EMBL/Gene Bank, Accession No J03279): SEQ ID No 1: 5'- GGA CGT AAG CAT CTC TGA AGT G -3' (22mer, TM=66°C) SOD-2 (downstream primer, region 1018-1036 of the sequence entered in EMBL/Gene Bank, accession n° J03279). SEQ ID NO 2: 5'- GCC GTC GAC GGA CCC CTC AAG ACC CCT C (28mer, TM=64°C)) The SOD-2 primer has a matching region of 19 bases of length (TM=64°C) and a 5'-non- matching region which carries a BamHI restriction site. DNA amplification was performed in 50 mM KC1, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl 2 , 500 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), 0.5 mM of each primer, 50 or 100 ng of genomic DNA and 2.5 Units of Taq DNA polymerase (Perkin- Elmer Corp.) in 100 ml of reaction volume. Time and temperature used in each amplification stage was as follows: 1 minute at 94°C for the denaturation, 1.5 minutes at 63°C for the annealing and 2 minutes for the elongation. The PCR reaction generated a DNA fragment of 965 base pairs (bp) of length, seen in agarose gel electrophoresis, which may include the entire coding sequence of the SOD gene of 462 bp, an upstream sequence of 331 bp and a downstream sequence of 172 bp. The second round (semi-nested PCR) was performed using the primer SOD-3 and a third internal primer SOD-4 which spans the region 971-991 of the sequence entered in EMBL/Gene Bank, accession n° J03279 and has the following sequence: SEQ ID NO 3: 5'- GCC GTC GAC ACA CTT GGT GAA TGATCA AGG-3'. Primer SOD-4 has a matching region of 21 bases of length (TM=60°C) and a 5'-non- matching region which carries a Sail restriction site. The semi-nested PCR reaction, performed with the above conditions unless the annealing temperature of 60°C, generated a shorter DNA fragment of 920 base pairs (bp) of length which may include the entire coding sequence of the SOD gene of 462 bp, an upstream sequence of 331 bp and a downstream sequence of 127 bp,. Example 3: Subcloning of the Cu/Zn SOD gene into the plasmid vector pCRH This example relates to subcloning of the Cu/Zn SOD gene into the plasmid vector pCRII. Following amplification, the products of the reactions were loaded on a 1.2 % low-melting temperature agarose gel and run on an electrophoresis apparatus (MiniSubgel DNA cell, BIO-RAD Laboratories, Inc. Hercules, CA, USA,). The appropriate DNA band was then isolated from the agarose gel through standard methods and purified by phenol extraction (Sambrook J., Fritsch E.F. and Maniatis T. Molecular Cloning. A manual laboratory, Cold Spring Harbor Laboratory, New York.1989). The purified DNA fragment was finally inserted, by ligation with the enzyme T4 DNA ligase at 12 °C for 16 hours, in the multiple cloning site (MCS) of the linearized plasmid pCRII of 3932 bp of length using the TA Cloning System (Invitrogen Corp. San Diego, CA). The pCRII vector is a cloning vector which contains single 3' deoxythymidylate overhangs to allow direct ligation of PCR products, and both ampicillin and kanamycin resistence genes for its selection in E.coli cells. The construct (i.e. plasmid pCRII plus the insert) was then transfected and replicated in E. coli cells HB 101. Finally, the plasmid DNA was extracted from bacterial cells by the alkaline lysis method of Bimboim and Doly ( Bimboim H.C., J. Doly. 1979 Nucleic Acids Research. 7:1513) and purified by Nucleobond AX-100 cartridges (Macherey-Nagel GmbH, Duren, Germany). Example 4: Construction of plasmid pEMBL-SOD 374 The construct was prepared by using the pEMBLyex4 plasmid as a vector in which the previously isolated locus containing the SOD gene, the downstream and the modified upstream region were cloned. The construct was named pEMBL-SOD 374 and is shown in Fig 2. It is a construct in which the subcloned fragment carries the coding region of the SOD gene (462 bp) and an upstream region of 374 bp. To perform such a construct, the fragment which carries the Cu/Zn SOD gene, previously subcloned in pCRII plasmid, was directly excised by the restriction enzymes BamHI and Sail. The above enzymatic digestion produced a Bam Hl-Sall 963 bp fragment which was purified by gel electrophoresis and subcloned in the BamHI-Sall sites of the vector pEMBLyex4. Example 5: Preparation of yeast strains X4004 and GRF18 transformed with pEMBL-SOD 374 The expression of the yeast Cu/Zn-SOD gene from the plasmid pEMBL-SOD 374 was tested following its insertion in two different strains of S. Cerevisiae which are typically frequently utilized for the expression of homologous or heterologous genes in yeasts (Martegani et al., Appl. Microbiol. Biotechnology. 37: 604-608 (1992), Alberghina L. et al., Biotechnology and Applied Biochemistry. 14:82-92 (1991), Pradyumna K. et al., Biotechnology and Bioeng. 40:235-246 (1992), Yong Soo Park et al., Biotechnology and Bioeng. 41 :854-861 (1993), Scott, et al., Biotechnology and Bioeng. 41:801-810 (1993), Jih-Han Hsieh et al., Biotechnology and Bioeng. 32: 334-340 (1988)). The following haploid strains were used: - X4004, whose genotype is: MATa/ lys5/ ura3/ met2/ trpl, and - GRF18, whose genotype is: MATa/ leu2-3, 112/ His3-11,15. These strains can be fermented at high biomass quite efficiently in selective or semisynthetic media due to the markers present on the plasmid vector pEMBLyex4 (LEU2-d and URA3) which complements respectively the leucine auxotrophy on the Ieu2-GRF18 strain (the GRF18 strain carries a leu 2-3,112 double frameshift mutation that reverts extremely rarely) and the uracil auxotrophy of the ura3-X4004 strain. Naturally, following insertion of the plasmid, the novel strains loose those specific auxotrophies. GRF18, carrying the novel plasmid will still be auxotroph for histidine, while X4004, carrying the novel plasmid will still be auxotroph for methionine and tryptophane. The yeast transformation with the plasmid constructs were performed as follows. Before transformation: yeast strains (X4004 or GRF18) plated on "petri-plates" as a streak, - dissolve a few cells scraped from the plate by a loop into 10 ml of sterile distilled water, sonicate for 5" and count by Coulter Counter (O.D600 under 0.1), inoculate about 4x10 7 total cells in 200 ml (to have 2-3 xlO 5 cells/ml, about 0.6 - OD600) of YEPD medium (in an 1 liter flask), - grow for 16 hours at 30°C under mild shaking (about 6 generation) until about - lxl 0 7 cells/ml (OD600= 2-3). YEPD complete medium for routine growth of the cells before transformation was made as follows: 1% yeast extract, 2% peptone, 2% glucose, 2% Bacto agar (for petri-plates) and distilled water. Autoclave all components for 20 min. at 120°C. Transformation was performed as follows: A total of 2xl0 8 cells are used for a treatment (transformation of the strain with a 1 mg of plasmid DNA), e.g. a total of lxl 0 9 cells for 5 treatments (100 ml of culture broth containing lxlO 7 cells/ml). Transformation is carried out through the lithium chloride method of Ito modified by Schiestl and Gietz (R.H.Schiestl, R.D. Gietz, High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier, (1989), Current Genetics. 16:339-346). The transformants are plated on agar plates (synthetic minimal medium) lacking leucine (to select for plasmid-containing GRF18 cells) or lacking uracile (to select for plasmid-containig X4004 cells) and grown at 30°C. Single colonies are stored either at 4° C by streaking them on fresh selective plates or at -80°C in 15% glycerol. All the transformations were performed in double on the base of the following scheme: plasmid DNA Omg yeast strain (2xl0 8 cells) pEMBL-SOD374 X4004 pEMBLyex4 X4004 (negative control) pEMBL-SOD 374 GRF18 pEMBLyex4 GRF 18 (negative control) Strain GRF 18 was grown on a synthetic medium without the amino acid leucine for the selection of plasmid-bearing strains, while in the case of X4004, the strain is grown on a synthetic medium without uracile. The induction of the expression in both cases was performed by shifting from minimal medium containing glucose to medium containing galactose as carbon source as follows: The transformants, following streaking on plates, are grown in 50 ml of synthetic minimal medium (lacking leucine for transformed GRF 18 or uracile for transformed X4004) containing 2% of glucose, at 30°C for 12-14 hours under mild shaking and aeration to reach about 2,5-3 x 10 7 cells/ml (O.D600 = 4-5). 50 ml of selective synthetic medium containing 2% of galactose are inoculated with 5x10 7 cells (2 ml of preculture) to reach about 1x10^ cells/ml. Growth was for about 17-18 hours (about 7 generations) at 30°C under mild shaking and aeration to reach about 2-3 xlO 7 cells/ml (O.D600 = 4-5). 2xl0 8 cells (e.g. about 10 ml of culture) are removed and used for protein extraction. The medium to grow transformed cells was made as follows: Synthetic selective medium (to grow transformed X4004 cells): 2% of carbon source (glucose or galactose), 2% Bacto agar (for petri-plates), 50 mg/liter of L-lysineHCl, 50 mg/liter of L-methionine, and distilled water. Autoclave components for 20 min. at 120°C, add difco yeast nitrogen base (YNB) without amino acids (filtered concentrated stock solution 10X (67 g/liter)), 50 mg/ml of L-tryptophan (filtered concentrated stock solution); Synthetic selective medium (to grow transformed GRF 18 cells): 2% of carbon source (glucose or galactose), 2% Bacto agar (for petri-plates), 50 mg/liter of L-histidine, distilled water. Autoclave components for 20 min. at 120°C, add difco yeast nitrogen base (YNB) without amino acids (filtered concentrated stock solution 10X (67 g/liter)). The ability of a microbial strain to produce a given polypeptide may be tested through several approaches. Initially, it is advisible to evaluate the productivity of the simple polypeptidic chain. In fact, the first question that has to be answered is whether the expression machinery of the novel cell is working efficiently in relation to the biosynthesis of the desired chemical entity. Thus, to test the presence of large amounts of a chemical entity like a polypeptide in cell extracts, the total protein extracts are first run on SDS denaturing polyacrylamide gels which discriminate according to the molecular weight of the polypeptidic chain. In addition, since it can be disposed of "isogenic" yeast strains (see negative controls), which do not carry the SOD containing-expression plasmid, the electrophoretic profile of a cell extract obtained from the plasmid-bearing strain may be compared with the cell extract obtained from a traditional strain. Knowing the molecular weight of SOD (the SOD polypeptide chain is composed of 154 amino acids with a corresponding molecular weigth of 15700 Daltons), the two profiles for the presence in the region of the gel corresponding to the above molecular weight of a SOD band have been compared. The visualization was performed through non-specific staining with Coomassie-blue. Analytical techniques used to evaluate the productivity of the clones was shown by SDS- polyacrylamide electrophoresis as follows: a) Protein yeast extraction: The protocol that was used for preparation of total protein extracts from yeast, partially modified from the method of Jazwinski (S. Michail Jazwinski, 1990, Methods in Enzymology, vol.182 p. 154) was as follows: 2xl0 8 cells are concentrated (in a 15 ml falcon tube) by centrifugation (4000 rpm at 4°C, 5 min.), wash pellet by sterile water at 4°C (transfer to eppendorf vials), - resuspend the pellet in 400 microliters of IX PBS buffer, add 400 microliters of glass microbeads (prechilled at -20°), add 4 microliters (1 mg/ml) of pepstatin (proteases inibitor), vortex for 3 minutes (twice on ice), store 10 microliters of supernatant at -20°C for protein assay. The staining with Coomassie blue of the gel shows a clear electrophoretic band in the samples (GRF 18 cells transformed with plasmid pEMBL-SOD 374), corresponding to molecular weigth of the Cu/Zn SOD yeast protein loaded on the same gel. Such bands were not observed in the negative samples control (GRF 18 cells transformed with the plasmid pEMBLyex4 which does not carry the SOD gene and X4004 cells transformed with the plasmid pEMBLyex4 which does not carry the SOD gene). Approximately the same quantity of protein extracts for each sample (about 1 mg) was loaded on the polyacrylamide gel. b) SOD activity determination by PlU-test The expression of a homologous or heterologous enzyme can be determined by an appropriate activity test. Therefore, the evaluation of the expression of yeast cells transformed by the two molecular constructs was performed by quantification of the SOD activity after induction of the cultures. The growth of the cultures was monitored by determining cell numbers with a counter (Coulter counter ZBI) (Lotti et al., Appl. Microbiol. Biotechnol. 28: 160-165 (1988)) or with measurement of absorbance at 600 nm. The protein extracts were prepared as described under „protein yeast extraction". The presence of Cu/Zn SOD activity in total yeast cells extracts was detected by the method of Marklund and Marklund, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol (Marklund S. and Marklund G., Eur. J. Biochem. 47: 469-474 (1974)). The expression experiments were performed as batch fermentation in complete synthetic medium:. Complete synthetic medium (Sherman F., Methods in Enzymology. vol 194, Academic Press) with and without copper (0.0025 g/liter) and zinc (0.05 g/liter) was performed as follows (in grams per litre): Bacto yeast Nitrogen base w/o amino acids (Difco Laboratories, Detroit, MI), 6.7; carbon source (glucose or galactose), 20; Adenine sulfate, 50; uracil, 50; L-tryptophan, 50; L-histidine, 50; L-arginine-HCl, 50; L-methionine, 50; L- tyrosine, 50; L-isoleucine, 50; L-Lysine-HCl, 50; L-phenylalanine, 50; L-glutamic acid, 50; L-aspartic acid, 50; L-valine, 50; L-threonine, 50; L-serine, 50. Table 1 shows SOD activity data (in strain GRF 18) upon expression of the SOD gene before deletion of MCS and bacterial sequences of three experiments and the mean values: Table 1 The average expression values are 301 PlU/ml for the vector pEMBL-SOD 374, and 13 PlU/ml for the vector pEMBLyex4 (without the insert). These values are important to judge the specific expression of the target gene. It is determined in standardized laboratory batch fermentations. The results show on average a 23 times elevated expression level compared to the original strain (GRF 18 with plasmid but without insert). Compared to a purchased wine yeast strain ("Seccoferm") a 22 time increase in expression was observed in the laboratory batch assays. It is shown that the economy of the manufacturing process is highly increased by the new constructions. Example 6: Preparation of the vector pEMBL-SOD 374 w/o MCS From the experiments made in Example 5 the conclusion can be taken that the performance of the yeast strain GRF 18 which contains the molecular construct pEMBL-SOD 374 is very high. As control served the same strains which do not contain any construct. Thus, the molecular construct pEMBL-SOD 374 was used to built the final expression system by deleting any non-yeast sequence. The synthetic sequence of the "multiple cloning site" was completely deleted by a double cutting at both ends of the polylinker with the enzymes Sst I and Hind III. This operation permitted the excision of the entire 95 bases of the artificial sequence from the molecular construct pEMBL-SOD 374 and allowed to insert the complete yeast Cu/Zn SOD gene between the remaining natural sites. Detailed protocol: 1) The clone pEMBL-SOD 374 was cut by the enzymes Hind III (New England Biolabs Inc., USA) and Sac I (New England Biolabs Inc., USA) to excise the polylinker from the rest of the vector. The enzyme Hind III recognizes the unique site "A/AGCTT" at one end of the multiple cloning site, while the enzyme Sad is a isoschizomer of the enzyme SstI and recognizes the same unique sequence "GAGCT/C" at the other end of the multiple cloning site. Following this double cutting, a fragment Hind Ill-Sac I of 8705 bases which carries all the yeast and bacterial sequences of the vector pEMBL without the polylinker, was isolated from agarose gel electrophoresis by electroelution. The 8705 bp-fragment was finally purified by phenol-chloroform treatment and concentrated by ethanol precipitation. 2) The ends of the 8705 bp Hind Ill-Sac I fragment were treated with the enzyme Polymerase I "Klenow fragment" to create compatible ends on the fragment for further manipulations. This treatment blunts both ends of the fragment by filling of the Hind III end (which is a 5' protuding termini) and cutting of the Sac I end (which is a 3' protuding termini). The blunt-ended fragment was finally purified by phenol-chloroform and ethanol precipitation. 3) The DNA fragment carrying the yeast Cu/Zn SOD gene and being present on the clone pEMBL-SOD 374 was cut with the enzymes Eco RI (New England Biolabs Inc., USA) and Sal I (New England Biolabs Inc., USA) which recognize respectively the sites "G/AATTC" and "G/TCGAC". This double cutting permits to isolate a fragment of 916 bp which carries a 342 bp yeast upstream sequence, the entire open reading frame of the yeast Cu/Zn SOD gene of 462 bp and a yeast downstream sequence of 112 bp. This 916 bp fragment, which does not carry any other bacterial or artificial sequence, was isolated from agarose gel electrophoresis by electroelution and purified by phenol-chloroform treatment and ethanol precipitation. 4) After purification, the Eco Rl-Sal I fragment was treated with the enzyme Polymerase I "Klenow fragment" which performs the filling of the Eco RI and Sal I ends (which are both 5' protuding termini) to make them compatible to be subcloned to the ends of the purified pEMBL vector from which the polylinker was previously deleted. The blunt- ended fragment was again purified by phenol-chloroform and ethanol precipitation. 5) The blunt-ended 916 bp fragment containing the yeast Cu/Zn SOD gene was finally subcloned into the blunt-ended 8705 bp fragment-pEMBL vector by ligation with the enzyme T4 DNA polymerase (Boehringer Mannheim GmbH, Mannheim). The ligation was performed at 16°C for 20 hours. 6) After transformation of the ligation mix (blunt-ended 916 bp fragment plus blunt- ended 8705 bp fragment) in E. Coli bacterial cells (XL 1 -Blue strain), a random screening was performed to isolate the clones which carry the right sequences. A number of bacterial clones from the transformation were grown over night at 37°C in LB broth medium and the DNA plasmid from the clones was extracted by "mini preps" method ("Wizard" minipreps DNA purification system, Promega Corp., USA). PCR amplification as well as DNA sequencing were used to verify both the presence and the right orientation of the fragment containing the yeast Cu/Zn SOD gene in the clones. The correctness was confirmed by sequencing and agarose gel electrophoresis. Example 7: Preparation of the final vector pEMBL-SOD w/o MCS w/o Coli The clone "pEMBL-SOD 374 w/o MCS" obtained from Example 6 carries only the yeast sequences (yeast funtional sequences and yeast Cu/Zn SOD gene) and the bacterial sequences. The non-yeast part of the "pEMBL-SOD 374 w/o MCS" clone carries the origin of replication for the bacterium Escherichia Coli, the ampicilline bacterial selectable marker and the origin of replication derived from the filamentous bacteriophage fl . This region covers more than 4000 bases and must be deleted by enzymatic digestion in order to obtain a final vector which carries only yeast sequences. The final vector following such a manipulation will be able to replicate only in yeast strains. In fact, the only sequences present in the final vector are: Leu 2-d yeast selectable marker useful to select the yeast cell transformants, the origin of replication of 2-micron yeast episome that provides high mitotic stability and replication of the plasmid, the entire yeast expression hybrid UAS GAL/CYC system that provides high level of transcription of the gene under its control, the yeast SOD gene (with upstream and downstream functional yeast sequences) which produces the enzyme Cu/Zn Superoxide Dismutase. Detailed protocol for preparation of vector: 1) The clone "pEMBL-SOD 374 w/o MCS", previously deleted from the multiple cloning site, was cut with the blunt endonucleases Stu I (in position 5942 of the original pEMBLyex4 vector) and Nru I (in position 1791 of the original pEMBLyex4 vector). The enzyme Stu I (Pharmacia, Uppsala) recognizes the unique blunted site "AGG/CCT" and the enzyme Nru I (Pharmacia, Uppsala) recognizes the unique blunted site "TCG/CGA". The resulting Stu I-Nru I fragment of 4689 bp carrying all the bacterial sequences was separated from the rest of the pEMBL vector by gel electrophoresis. The double cutting deletes also a part of the yeast selectable marker URA 3 on the vector but it preserves the complete function of the other yeast selectable marker LEU-2d, which can be used for the selection of the clones during the further manipulations. 2) The fragment pEMBL vector containing only yeast sequences and the yeast Cu/Zn SOD gene were isolated from agarose gel by electroelution and purified by phenol- chloroform and ethanol precipitation. 3) The ends of the purified pEMBL vector (both ends are blunted) were rejoined together by the enzyme T4 DNA ligase (Boehringer Mannheim GmbH, Mannheim) and the ligation mix was used to transform GRF 18 yeast strain. 4) Clones from transformation were plated on agar plates with synthetic minimal medium lacking leucine for the selection (by the selectable marker Leu 2-d) of the GRF 18 cells containing the deleted plasmid. 5) A number of yeast colonies obtained from the transformation were tested by PCR to confirm both the presence of the yeast fragment containing the yeast Cu/Zn SOD gene and the absence of any bacterial sequence between the Stu I and Nru I enzymatic sites. To confirm the presence of the yeast fragment on the final construct "pEMBL-SOD w/o MCS w/o Coli", PCR amplification was performed with the two flanking primers SOD proA and SOD proB. A putative electrophoretic band of about 1133 bp length confirmed the presence of the fragment containing the yeast Cu/Zn SOD gene in six yeast clones. The correctness was confirmed by sequencing and agarose gel electrophoresis. The final vector "pEMBL-SOD w/o MCS w/o Coli" derived from such manipulations does not carry any bacterial or artificial sequence and will be able to replicate only in yeast strains because of the presence of yeast sequences only. The final vector is presented as restriction map in FIG. 3. Example 8: Evaluation of the expression of the yeast Cu/Zn SOD gene in the new yeast strain This example relates to the evaluation of the expression of the yeast Cu/Zn SOD gene in the new yeast strains as constructed in Examples 6 and 7. All the experiments were performed as in Example 5 with growing yeast cells in complete synthetic media. Table 2 shows the presence of SOD activity in total yeast cell extracts upon expression of SOD gene after deletion of MCS and bacterial sequences in GRF 18-pEMBL SOD 374 in complete synthetic medium: Table 2. The test results indicate that the vector pEMBL-SODw/oMCSw/oColi expresses the yeast Cu/Zn SOD gene in higher yield compared to vector pEMBLyex4 (in GRF 18). Example 9: Isolation and purification of yeast Cu/Zn SOD The production of the target polypeptide, such as the yeast superoxide dismutase, is performed under aerobic conditions in computer-controlled fermenters, e. g. according to Alberghina et al. ibid. Controll parameters of fermentation are temperature, dissoluted oxygen, pH and ethanol concentration. The purification of the polypeptide comprises the following steps: 1. The yeast cells in the fermentation broth is directly lysed by homogenization with a homogenizer, e. g. an APV Gaulin, at a temperature of between 20° and 30°C, a pressure of between 600 and 800 bar and 3 cyles, or with a dynobed mill, e. g. a Dyno Mill Model KDL filled with acid washed 0.3 mm diameter, and recirculating the suspension through the mill at 160 ml/min for 1-2 min at room temperature. 2. The separation of cell debris and proteins is performed by centrifugation, e. g. with a Beckmann J2-21 centrifuge with JA-10 fixed angle rotor for 60 min at a speed of 14'OOOg, by or microfiltration, e. g. by tangential flow filtration with Minitan (Millipore Corp., Bedford) equipped with a 0.45 μm cut-off membrane. 3. Concentration and buffer change by ultrafiltration and diafiltration: Condition: Tangential flow filtration with Minitan (Millipore Corp., Bedford) equipped with a lO'OOO Dalton cut-off cellulose membrane (PLGCOMP 04 membrane). Buffer: 20 mM Tris-HCl pH 8.0. 4. Purification by cationic exchange chromatography, e. g. with DEAE-Sepharose. Conditions: Loading buffer: 20 mM Tris-HCl pH 8.0; elution buffer: 20 mM Tris-HCl pH 8.0, 1 M NaCl. 5. Concentration and buffer change by ultrafiltration and diafiltration to obtain the final buffered SOD solution. Condition: Tangential flow filtration with Minitan (Millipore Corp., Bedford) equipped with a lO'OOO Dalton cut-off membrane. Buffer: 20 mM Tris-HCl pH 8.0. At the end of fermentation the activity is at least 5000 PlU/ml. The yield obtainable with the new strain according to the invention is at least 10 times higher compared to the yield obtainable with purchasable bakers yeast. From 1 g bakers yeast 5000 PIU can be isolated, whereas at least 40O00 PIU can be isolated from 1 g pEMBL-SOD 374 GRF 18 yeast. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: PENTAPHARM AG (B) STREET: ENGELGASSE 109 (C) CITY: BASEL (D) STATE: BS (E) COUNTRY: SWITZERLAND (F) POSTAL CODE (ZIP): CH-4002 (G) TELEPHONE: xx41-61-7064848 (H) TELEFAX: xx41-61-3199619 (ii) TITLE OF INVENTION: IMPROVED PRODUCTION OF POLYPEPTIDES IN YEAST (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (B) STRAIN: S288C (vii) IMMEDIATE SOURCE: (A) LIBRARY: EMBL No J03279 (viii) POSITION IN GENOME: (B) MAP POSITION: 81-102 (C) UNITS: bp (ix) FEATURE: (A) NAME/KEY: miscjeature (B) LOCATION: 1..22 (ix) FEATURE: (A) NAME/KEY: primer_bind (B) LOCATION: 1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GGACGTAAGC ATCTCTGAAG TG 22 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (B) STRAIN: S288C (vii) IMMEDIATE SOURCE: (A) LIBRARY: EMBL No J03279 (viii) POSITION IN GENOME: (B) MAP POSITION: 1018-1036 (C) UNITS: bp (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1. 28 (ix) FEATURE: (A) NAME/KEY: primer_bind (B) LOCATION: 1..28 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCCGTCGACG GACCCCTCAA GACCCCTC 28 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (B) STRAIN: S288C (viii) POSITION IN GENOME: (C) UNITS: bp (ix) FEATURE: (A) NAME/KEY: miscjeature (B) LOCATION: 1..30 (ix) FEATURE: (A) NAME/KEY: primer_bind (B) LOCATION:!..30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCCGTCGACA CACTTGGTGA ATGATCAAGG 30

Claims: Claims: I . A new expression vector for the production of a polypeptide in yeast comprising a sequence coding for said polypeptide, and other sequences allowing expression of said polypeptide only in yeast, said other sequences lacking any non-yeast sequences. 2. A vector according to claim 1 wherein the sequence coding for said polypeptide is a yeast sequence under the control of a yeast promoter. 3. A vector according to claim 1 comprising a sequence coding for a yeast enzyme under the control of a yeast promoter. 4. A vector according to claim 1 which is a yeast plasmid comprising the Cu/Zn SOD gene which is under the control of the GAL/CYC promoter. 5. A vector according to claim 1 which is plasmid pEMBL-SODw/oMCS,w/oColi. 6. A vector according to claim 1 wherein the sequence coding for said polypeptide is a non-yeast sequence under the control of a yeast promoter. 7. A vector according to claim 6 wherein the sequence coding for said polypeptide is a human or animal sequence under the control of a yeast promoter. 8. A yeast strain comprising a vector according to claim 1. 9. A yeast strain according to claim 8 which is GRF 18 comprising plasmid pEMBL- SODw/oMCS,w/oColi. 10. A method for the production of a new expression vector acording to claim 1, characterized in that from a shuttle vector able to express a polypeptide in a yeast strain any non-yeast sequences are excised, and optionally the sequence coding for said polypeptide is replaced by a sequence coding for another polypeptide. I I . A method for the production of a yeast strain according to claim 8 characterized in that a yeast strain is transformed with a vector according to claim 1. 12. A method for the production of a polypeptide in a yeast strain comprising fermentation of a yeast strain according to claim 8 and isolating the polypeptide.

Inventors: SCHREIER THOMAS, VOEGELI RAINER, SCHREIER, THOMAS, VOEGELI, RAINER
Applicants: PENTAPHARM AG, SCHREIER THOMAS, VOEGELI RAINER, SCHREIER, THOMAS, VOEGELI, RAINER
Application year: 1997
Main classification: C12N15
IPC codes: C12N1, C12N9, C12N15, C12P21, C12R1