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Details for Patent: 4,801,542

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Details for Patent: 4,801,542

Title: Expression of biologically active PDGF analogs in eucaryotic cells
Abstract:Biologically active PDGF analogs expressed in eucaryotic cells 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. In particular, DNA sequences encoding polypeptides substantially homologous to the B chain of PDGF are preferred. A secretory signal sequence may be provided upstream of the gene, enabling secretion of the gene product from the 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:Feb 25, 1985
Application Number:06/705,175
Claims:1. A DNA construct capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promotor followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF, and a signal sequence positioned upstream from and in proper reading frame wih said gene, said signal sequence directing the secretion of the protein from the eucaryotic cell.

2. The DNA construct of claim 1 wherein the cultured eucaryotic cell is a cultured yeast cell.

3. The DNA construct of claim 2 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.

4. The vector of claim 2 wherein said gene is followed downstream by a triose phosphate isomerase terminator.

5. The vector of claim 1 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having biological activity.

6. The vector of claim 1 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having biological activity.

7. The DNA construct of claim 6 wherein the derivative of the v-sis gene of simian sarcoma virus is the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF.

8. The DNA construct of claim 1 wherein said gene is the human cDNA gene for PDGF or portions thereof encoding a protein having biological activity.

9. A DNA construct 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.

10. A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said DNA construct containing a transcriptional promoter 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, and a signal sequence directing the secretion of the protein from the eucaryotic cell.

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

introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF, and a signal sequence capable of directing the secretion of the protein from the eucaryotic cell;

growing said cultured eucaryotic cell in an appropriate medium; and

isolating the protein product of said gene from said cultured eucaryotic.

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

13. The method of claim 11 wherein the cultured eucaryotic cell is a cultured yeast cell.

14. The method of claim 13 wherein said promoter is the yeast alpha-factor promoter or the yeast triose phosphate isomerase promoter.

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

16. The method of claim 13 wherein said gene is followed downstream by a triose phosphate isomerase terminator.

17. The method of claim 11 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having biological activity.

18. The method of claim 11 wherein said said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having biological activity.

19. The method of claim 18 wherein the derivative of the v-sis gene of simian sarcoma virus is the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF.

20. The method of claim 11 wherein said gene is the human cDNA gene for PDGF or portions thereof encoding a protein having biological activity.

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

introducing into a cultured eucaryotic cell a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said DNA construct containing a transcriptional promoter 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, and a signal sequence directing the secretion of the protein from the eucaryotic cell;

growing said cultured eucaryotic cell in an appropriate medium; and

isolating the protein product of said gene from said cultured eucaryotic cell.

22. A cultured eucaryotic cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promotor followed downstream by a gene encoding a protein having substantially the same biological activity as PDGF, and a signal sequence capable of directing the secretion of the protein from the eucaryotic cell.

23. The cultured eucaryotic cell of claim 22 wherein the cultured eucaryotic cell is a cultured yeast cell.

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

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

26. The yeast cell of claim 23 wherein said gene is followed downstream by a triose phosphate isomerase terminator.

27. The yeast cell of claim 23 wherein said vector is the plasmid p117-2.

28. The yeast cell of claim 23 wherein said vector is the plasmid YEpVS.alpha..

29. The yeast cell of claim 23 wherein said vector is the plasmid YEpVS2.alpha..

30. The cultured eucaryotic cell of claim 22 wherein said gene is the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having biological activity.

31. The cultured eucaryotic cell of claim 22 wherein said gene is a derivative of the v-sis gene of simian sarcoma virus or portions thereof encoding a protein having biological activity.

32. The cultured eucaryotic cell of claim 22 wherein the derivative of the v-sis gene of simian sarcoma virus is the portion of the v-sis gene encoding a polypeptide which is substantially homologous to the B chain of PDGF.

33. The cultured eucaryotic cell of claim 22 wherein said gene is the human cDNA gene for PDGF or portions thereof encoding a protein having biological activity.

34. The yeast cell of claim 23 wherein said vector is the plasmid pVSBm.

35. A cultured eucaryotic cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said DNA construct containing a transcriptional promoter 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, and a signal sequence directing the secretion of the protein from the eucaryotic cell.

36. The plasmid pVSB.

37. The plasmid pVSBm.
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