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United States Patent No. 5,854,018

Issue Date: December 29, 1998

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 yeast strain capable of expressing a polypeptide ordinarily exogenous to yeast, comprising transforming said yeast strain with a DNA transfer vector that comprises:

   a.   bacterial and yeast origins of replication and genes for phenotypic selection of both bacterial and yeast moieties transformed with said vector;
   b.   a DNA fragment comprising a structural gene encoding said polypeptide;
   c.   a yeast promoter genetically distinct from said structural gene obtained from the DNA sequence 5' flanking the start signal of a yeast structural gene; and
   d.   translation start and stop signals for said structural gene encoding said polypeptide that are positioned so that said structural gene encoding said polypeptide is under the control of said promoter while maintaining said origins of replications and genes for phenotypic selection.

2. The process of Claim 1, wherein the vector further comprises a transcription terminator located between the 3' end of a coding strand of said structural gene encoding said polypeptide and said origins of replication.
3. The process of Claim 1, wherein the DNA fragment comprising said structural gene further comprises a translation start signal and a translation stop signal.
4. The process of Claim 1, wherein the gene for phenotypic selection of bacteria encodes a polypeptide that confers antibiotic-resistance and wherein the gene for phenotypic selection of yeast encodes a polypeptide that complements a mutation carried by the yeast strain to be transformed.
5. The process of Claim 4, wherein the yeast strain is Saccharomyces cerevisiae.
6. The process of Claim 5, wherein the yeast strain is strain RE 218.
7. The, process of Claim 1, wherein the amino acid sequence of said polypeptide is selected to correspond to the amino acid sequence of a polypeptide 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.
8. The process of Claim 1, wherein the polypeptide is the A or B chain of human insulin.
9. The process of Claim 1, wherein the yeast promoter includes a transcription start site of the yeast structural gene.
10. A process of producing a polypeptide comprising culturing a yeast strain transformed by the process of Claim 1, and recovering said polypeptide.
11. The process of Claim 10, wherein the amino acid sequence of said polypeptide is an intermediate from which the A and B chains of human insulin can be obtained by cleavage away of superfluous amino acids.
12. A DNA expression vector, capable in a transformant strain of yeast of expressing a polypeptide ordinarily exogenous to yeast, said vector comprising bacterial and yeast origins of replication and a yeast promoter from the DNA sequence 5' flanking the start signal of a yeast structural gene, said yeast promoter being capable of directing the expression of a gene encoding said polypeptide.
13. The DNA expression vector of Claim 12, which further comprises a gene encoding a polypeptide under the control of the yeast promoter.
14. The DNA expression vector of Claim 13, wherein the promoter is from within the 1500 bp DNA sequence 5' flanking the start signal of a yeast structural gene, the promoter is resected free of the yeast structural gene and the start signal of the yeast structural gene, and the gene encoding the polypeptide is located at the endpoint of the resection.
15. The DNA expression vector of Claim 13, which further comprises a transcription terminator following the gene encoding the polypeptide in the 3' direction in which the gene is transcribed.
16. The DNA expression vector of Claim 13, which further comprises a gene encoding a polypeptide for phenotypic selection of a yeast transformant.
17. The DNA expression vector of Claim 16, which further comprises a gene for encoding a polypeptide for phenotypic selection in bacteria.
18. The DNA expression vector of Claim 17, wherein the gene encoding the polypeptide for phenotypic selection in bacteria comprises a gene encoding a polypeptide that confers antibiotic resistance and the gene encoding the polypeptide for phenotypic selection of the yeast transformant comprises a gene encoding a polypeptide that complements a mutation carried by the yeast strain to be transformed with the expression vector.
19. The DNA expression vector of Claim 13, wherein the polypeptide comprises the amino acid sequence of a polypeptide having the activity of a polypeptide 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.
20. The DNA expression vector of Claim 13, wherein the polypeptide is the A or B chain of human insulin.
21. The DNA expression vector of Claim 13, wherein the polypeptide is a mammalian polypeptide.
22. The DNA expression vector of Claim 15, wherein the polypeptide is a mammalian polypeptide.
23. The DNA expression vector of Claim 13, wherein the yeast promoter comprises a promoter for the yeast 3-phosphoglycerate kinase, glyceraldehyde-3 -phosphate dehydrogenase, or alcohol dehydrogenase structural genes.
24. The DNA expression vector of Claim 13, wherein the promoter is a yeast alcohol dehydrogenase promoter.
25. The DNA expression vector of Claim 13, wherein the origin of replication is the 2 micron yeast plasmid origin of replication.
26. The DNA expression vector of Claim 13, wherein the structural gene is a yeast chromosomal gene.
27. The DNA expression vector of Claim 13, wherein the yeast promoter further comprises a yeast transcription start site.
28. A process of producing a polypeptide comprising transforming a yeast strain with the DNA expression vector of Claim 12, culturing the transformed yeast strain, and recovering said polypeptide.
29. A yeast strain comprising a gene encoding a polypeptide ordinarily exogenous to yeast under the control of a yeast promoter from the DNA sequence 5' flanking the start signal of a yeast structural gene.
30. The yeast strain of Claim 29, wherein the yeast promoter is resected free of the yeast structural gene and the gene encoding the polypeptide is located at the endpoint of the resection.
31. The yeast strain of Claim 29, wherein the polypeptide has the amino acid sequence of a polypeptide having the activity of a polypeptide 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.
32. The yeast strain of Claim 29, wherein the polypeptide is the A or B chain of human insulin.
33. The yeast strain of Claim 29, wherein the polypeptide is a mammalian polypeptide.

34. A yeast strain capable of expressing a polypeptide ordinarily exogenous to yeast produced by a process comprising transforming said yeast strain with a DNA transfer vector comprising:

    a.   a bacterial and yeast origins of replication and genes encoding polypeptides for phenotypic selection of both bacterial and yeast moieties transformed with said vector;
    b.   a DNA fragment comprising a structural gene encoding said polypeptide;
    c.   a DNA fragment comprising a yeast promoter from the DNA sequence S' flanking the start signal of a yeast structural gene;
    d.   translation start and stop signals for said structural gene encoding said polypeptide positioned such that said structural gene for said polypeptide is under the control of said yeast promoter, while maintaining said origins of replication and genes encoding polypeptides for phenotypic selection.

35. The yeast strain of Claim 34, wherein said yeast promoter is resected free of said yeast structural gene and the gene encoding said polypeptide is located at the endpoint of the resection.
36. The yeast strain of Claim 34, wherein said polypeptide comprises the amino acid sequence of a polypeptide having the activity of a polypeptide 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.
37. The yeast strain of Claim 34, wherein the yeast promoter further comprises a yeast transcription start site.
38. A process of producing a polypeptide comprising culturing the yeast strain of Claim 34, 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