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United States Patent No. 5,618,676

Issue Date: April 8, 1997

EXPRESSION OF POLYPEPTIDES IN YEAST

Inventors:

Ronald A. Hitzeman
Franklin E. Hagie, IV
Benjamin D. Hall
Gustav Ammerer

Assignees:
Genentech, Inc.
Washington Research Foundation

Abstract:
A DNA expression vector capable, in a transformant strain of yeast, of expressing a biologically competent polypeptide ordinarily exogenous to yeast under the control of a genetically distinct yeast promoter, the polypeptide not being required for the growth of the transformant; the process of forming the transformant strain of yeast; and the transformant strain of yeast.

ISSUED CLAIMS

1. A process of forming a transformant of a given yeast strain, which transformant is capable of expressing a biocompetent polypeptide ordinarily exogenous to yeast and not required for growth of said strain. which comprises:
   
   a.  providing a DNA transfer vector having bacterial and yeast origins of replication and genes for phenotypic   selection of both bacterial and yeast moieties transformed with said genes;
   b.  providing a DNA fragment comprising a structural gene encoding said biocompetent polypeptide;
   c.  providing a DNA fragment comprising a yeast promoter genetically distinct from said structural gene which yeast promoter is from within the about 1500 bp DNA sequence 5' flanking the start codon of a yeast structural gene;
   d.  inserting the fragments of steps (b) and (c) into said transfer vector together with appropriately positioned translation start and stop signals for said structural gene for said biocompetent polypeptide to form an expression vector in which said structural gene for said biocompetent polypeptide is under the control of said promoter. while maintaining said origins of replications and genes for phenotypic selection, wherein the promoter is resected free of the yeast structural gene and the gene encoding the polypeptide is located at the endpoint of the resection; and
   e.  transforming said strain with the resulting expression vector, wherein said yeast strain is Saccharomyces cerevisiae.
2. The process of claim 1, which additionally comprises provision of a transcription terminator between the 3' end of said coding strand and said origins of replication in the direction in which the structural gene is transcribed.
3. The process of claim 1, wherein the DNA fragment of step (b) comprises translation start and stop codons.
4. The process of claim 1, wherein the bacterial selection phenotype is antibiotic resistance and wherein the yeast selection phenotype complements a mutation carried by the yeast strain to be transformed.
5. The process of claim 4, wherein the yeast strain transformed is strain RH 218.
6. The process of claim 1, wherein the amino acid sequence of said polypeptide is selected to correspond to the amino acid sequences of polypeptides selected from the group consisting of the normal and hybrid human interferons, human proinsulin, the A and B chains of human insulin, human growth hormone, somatostatin, and thymosin alpha 1.
7. The process of claim 1, wherein the yeast promoter includes a transcription start site of the yeast structural gene.
8. A method of producing a biocompetent polypeptide ordinarily exogenous to yeast comprising culturing a yeast strain transformed by the process of claim I and recovering said polypeptide.
9. A DNA expression vector, capable in a transformant strain of Saccharomyces cerevisiae of expressing a biologically competent polypeptide ordinarily exogenous to yeast and not required for growth of said strain, comprising a gene encoding a biologically competent polypeptide ordinarily exogenous to yeast under the control of a genetically distinct yeast promoter from within the 1500 bp DNA sequence 5' flanking the start codon of a yeast structural gene, wherein the promoter is resected free of the yeast structural gene and die gene encoding the polypeptide is located at the endpoint of the resection.
10. A DNA expression vector according to claim 9, wherein the polypeptide is not required for the growth of the transformant.
11. A DNA expression vector according to claim 9, comprising a transcription terminator following the gene encoding the polypeptide in the 3' direction in which the gene is transcribed.
12. A DNA expression vector according to claim 11, wherein the polypeptide is a mammalian polypeptide.
13. A DNA expression vector according to claim 11, comprising a gene for phenotypic selection of yeast transformants.
14. DNA expression vector according to claim 13, comprising a gene for phenotypic selection in bacteria.
15. A DNA expression vector according to claim 14, wherein the phenotypic basis for selection in bacteria is a gene encoding antibiotic resistance and wherein the phenotype for selection in yeast complements a mutation carried by a yeast strain suitable for transformation with the expression vector.
16. A DNA expression vector according to claim 9, wherein the amino acid sequence of the polypeptide is selected to correspond to the amino acid sequences of polypeptides selected from the group consisting of normal and hybrid human interferons, human proinsulin, the A and B chains of human insulin, human growth hormone, somatostatin, and thymosin alpha 1.
17. A DNA expression vector according to claim 9, wherein the polypeptide is a mammalian polypeptide.
18. A DNA expression vector according to claim 9, wherein the promoter is the promoter for the yeast 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, or alcohol dehydrogenase structural genes.
19. A DNA expression vector according to claim 9, wherein the promoter is a yeast alcohol dehydrogenase 15 promoter.
20. A DNA expression vector according to claim 9, comprising the yeast 2 micron plasmid origin of replication.
21. A DNA expression vector according to claim 9, wherein the yeast structural gene is a yeast chromosomal gene.
22. A DNA expression vector according to claim 9, comprising the yeast 2 micron plasmid origin of replication and wherein the promoter is that of a yeast chromosomal structural gene.
23. A DNA expression vector according to claim 9, wherein the yeast promoter includes a transcription start site of the yeast structural gene.
24. A method of producing a biocompetent polypeptide ordinarily exogenous to yeast comprising transforming a strain of Saccharomyces cerevisiae with a DNA expression vector of claim 9, and then culturing the yeast strain and recovering said polypeptide.
25. A Saccharomyces cerevisiae yeast strain comprising a gene encoding a biologically competent polypeptide ordinarily exogenous to yeast under the control of a genetically distinct yeast promoter from within the 1500 bp DNA sequence 5' flanking the start codon, of a yeast structural gene, wherein the promoter is resected free of the yeast structural gene and the gene encoding the polypeptide is located at the endpoint of the resection.
26. A yeast strain according to claim 25, wherein the amino acid sequence of the polypeptide is selected to correspond to the amino acid sequences of polypeptides selected from the group consisting of normal and hybrid 45 human interferons, human proinsulin, the A and B chains of human insulin, human growth hormone, somatostatin, and thymosin alpha 1.
27. A yeast strain according to claim 25, wherein the polypeptide is a mammalian polypeptide.
28. A yeast strain capable of expressing a biocompetent polypeptide ordinarily exogenous to yeast and not required for growth of said strain, which is produced by a process comprising:
    
    a.  providing a DNA transfer vector having bacterial and yeast origins of replication and genes for phenotypic selection of both bacterial and yeast moieties transformed with said genes;
    b.  providing a DNA fragment comprising a structural gene encoding said biocompetent polypeptide;
    c.  providing a DNA fragment comprising a yeast promoter genetically distinct from said structural gene which yeast promoter is from within the about 1500 bp DNA sequence 5' flank-in the start codon of a yeast structural gene;
    d.  inserting the fragments of steps (b) and (c) into said transfer vector together with appropriately positioned translation start and stop signals for said structural gene for said biocompetent polypeptide to form an expression vector in which said structural gene for said biocompetent polypeptide is under the control of said promoter, while maintaining said origins of replications and genes for phenotypic selection, wherein the promoter is resected free of the yeast structural gene and the gene encoding the polypeptide is located at the endpoint of the resection; and
    e.  transforming said strain with said expression vector, wherein said strain is Saccharomyces cerevisiae
    
29. A yeast strain according to claim 28, wherein the amino acid sequence of said polypeptide is selected to correspond to the amino acid sequences of polypeptides selected from the group consisting of the normal and hybrid human interferons, human proinsulin, the A and B chains of human insulin, human growth hormone, somatostatin, and thymosin alpha 1.
30. A yeast strain according to claim 28, wherein the yeast promoter includes a transcription start site of the yeast structural gene.
31. A method of producing a biocompetent polypeptide ordinarily exogenous to yeast comprising culturing a yeast strain of claim 28 and recovering said polypeptide.

For additional information, please contact:

Beth G. Etscheid, Ph.D.
Director of Licensing
Washington Research Foundation
2815 Eastlake Avenue E, Suite 300
Seattle, WA 98102
Tel: (206) 336.5600
Fax: (206) 336.5615
betschei@wrfseattle.org