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Details for Patent: 4,769,328

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Details for Patent: 4,769,328

Title: Expression of biologically active PDGF analogs in yeast
Abstract:Biologically active PDGF analogs expressed in yeast are disclosed. The analogs are produced by yeast strains transformed with an extrachromosomal element composed of a strong transcriptional promoter directing the expression of a gene which encodes a protein having substantially the same biological activity as PDGF. Suitable genes include the v-sis gene or a derivative of the v-sis gene of simian sarcoma virus or portions thereof, or the human cDNA gene for PDGF or portions thereof. A secretory signal sequence may be provided upstream of the gene, enabling secretion of the gene product from the yeast host cell. Mitogenic activity is one of the biological activities possessed by these PDGF analogs, making them useful in promoting the growth of mammalian cells.
Inventor(s): Murray; Mark J. (King County, WA), Kelly; James D. (King County, WA)
Assignee: ZymoGenetics Inc. (Seattle, WA)
Filing Date:Oct 12, 1984
Application Number:06/660,496
Claims:1. A vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.

2. The vector of claim 1 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.

3. The vector of claim 1 wherein said promoter is followed downstream by the signal sequence of the yeast mating pheromone alpha-factor.

4. The vector of claim 1 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof.

5. The vector of claim 1 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof.

6. The vector of claim 1 wherein said gene is the human cDNA gene for PDGF or portions thereof.

7. The vector of claim 1 wherein said gene is followed downstream by a triose phosphate isomerase terminator.

8. The vector of claim 6 wherein said human gene is derived from mRNA isolated from endothelial cells.

9. The vector of claim 1 wherein said protein is a disulfide-bonded dimeric protein.

10. A vector capable of replication in yeast and containing the yeast triose phosphate isomerase promoter, said yeast promoter being followed downstream by the signal sequence of the yeast mating pheromone alpha-factor, said signal sequence being followed downstream respectively by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, and a triose phosphate isomerase terminator.

11. A method of preparing biologically active PDGF analogs, comprising:

introducing into a yeast host a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF;

growing said yeast host in an appropriate medium; and

isolating the protein product of said gene from said yeast host.

12. The method of claim 11 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.

13. The method of claim 12 wherein said promoter is followed downstream by the signal sequence of the yeast mating pheromone alpha-factor.

14. The method of claim 13 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof.

15. The method of claim 14 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof.

16. The method of claim 15 wherein said gene is the human cDNA gene for PDGF or portions thereof.

17. The method of claim 16 wherein said human gene is derived from mRNA isolated from endothelial cells.

18. The method of claim 17 wherein said gene is followed downstream by a triose phosphate isomerase terminator.

19. The method of claim 11, including after isolation of said protein product, purifying said product by gel filtration, ion exchange chromatography of affinity chromatography.

20. The method of claim 19 wherein said medium need not contain antibiotics or heavy metals, and need not be depleted of specific nutrients.

21. The method of claim 19 wherein said protein is a disulfide-bonded dimeric protein.

22. A yeast cell transformed with a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.

23. The yeast cell of claim 22 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.

24. The yeast cell of claim 23 wherein said promoter is followed downstream by the signal sequence of the yeast mating pheromone alpha-factor.

25. The yeast cell of claim 24 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof.

26. The yeast cell of claim 25 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof.

27. The yeast cell of claim 26 wherein said gene is the human cDNA gene for PDGF or portions thereof.

28. The yeast cell of claim 27 wherein said human gene is derived from mRNA isolated from endothelial cells.

29. The yeast cell of claim 28 wherein said protein is a disulfide-bonded dimeric protein.

30. The yeast cell of claim 29 wherein said vector is the plasmid p117-2.

31. The yeast cell of claim 30 wherein said vector is the plasmid YEpVS.alpha..

32. The yeast cell of claim 31 wherein said vector is the plasmid YEpVS2.alpha..

33. The yeast cell of claim 32 wherein said gene is followed downstream by a triose phosphate isomerase terminator.

34. A method of promoting the growth of mammalian cells, comprising incubating the cells with a biologically active PDGF analog expressed by a yeast cell transformed with a vector capable of replication in yeast said vector containing a yeast promoter capable of directing the expression of the PDGF analog, said yeast promoter being followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF.

35. The method of claim 34 wherein said growth is carried out in vitro.

36. The method of claim 34 wherein said cells are normal cells or transformed cells.

37. The method of claim 34 wherein said protein is a disulfide-bonded dimeric protein.

38. The plasmid p117-2.

39. The plasmid YEpVS.alpha..

40. The plasmid YEpVS2.alpha..

41. A vector capable of replication in yeast and containing the yeast triose phosphate isomerase promoter, said yeast promoter being followed downstream by the signal sequence of the yeast mating pheromone alpha-factor, said signal sequence being followed downstream respectively by the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having substantially the same biological activity as PDGF, and a triose phosphate isomerase terminator.

42. The vector of claim 13 wherein said protein is a disulfide-bonded dimeric protein.

43. A vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF.

44. The vector of claim 15 wherein said protein is a disulfide-bonded dimeric protein.

45. A method of preparing biologically active PDGF analogs, comprising:

introducing into a yeast host a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF;

growing said yeast host in an appropriate medium; and

isolating the protein product of said gene from said yeast host.

46. The method of claim 32 wherein said protein is a disulfide-bonded dimeric protein.

47. A yeast cell transformed with a vector capable of replication in yeast and containing a yeast promoter capable of directing the expression of biologically active PDGF analogs, said yeast promoter being followed downstream by the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF, said portion encoding a protein having substantially the same biological activity as PDGF.

48. The yeast cell of claim 10 wherein said protein is a disulfide-bonded dimeric protein.
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