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Details for Patent: 4,766,073

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Details for Patent: 4,766,073

Title: Expression of biologically active PDGF analogs in eucaryotic cells
Abstract:Methods for expressing a variety of biologically active PDGF analogs in eucaryotic cells are disclosed. The methods generally comprise introducing into a eucaryotic host cell a DNA construct capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells. The DNA construct contains a transcriptional promoter followed downstream by a suitable DNA sequence. The DNA sequence may encode a protein substantially homologous to the A-chain or the B-chain of PDGF, or a portion thereof, or an A-B heterodimer. In addition, a portion of the DNA sequence may encode at least a portion of the A-chain, while another portion encodes at least a portion of the B-chain of PDGF. Eucaryotic cells transformed with these DNA constructs are also disclosed. Methods of promoting the growth of mammalian cells, comprising incubating the cells with a biologically active PDGF analog expressed by a eucaryotic host cell transformed with such a DNA construct, are also disclosed.
Inventor(s): Murray; Mark J. (Seattle, WA), Kelly; James D. (Seattle, WA)
Assignee: ZymoGenetics Inc. (Seattle, WA)
Filing Date:Aug 13, 1986
Application Number:06/896,485
Claims:1. 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 DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.

2. The vector of claim 1 wherein said eucaryotic cell is a yeast cell.

3. The vector of claim 1 wherein said protein includes at least one amino acid substitution of a cysteine residue.

4. 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 DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.

5. The vector of claim 4 wherein said eucaryotic cell is a yeast cell.

6. The vector of claim 4 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-109.

7. The vector of claim 4 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-97 fused in reading frame A-chain amino acids 92-104.

8. The vector of claim 4 wherein said protein includes at least one amino acid substitution of a crysteine residue.

9. 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 DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.

10. The vector of claim 9 wherein said eucaryotic cell is a yeast cell.

11. The vector of claim 9 wherein said protein is a monomeric species having biological activity.

12. The vector of claim 9 wherein said protein includes at least one amino acid substitution of a cysteine residue.

13. A vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.

14. 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 DNA sequence encoding a protein having substantially the same biological activity as PDGF, and a signal sequence positioned upstream from and in proper reading frame with said gene, said signal sequence directing the secretion of the protein from the eucaryotic cell.

15. 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 DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.

16. The method of claim 15 wherein said cultured eucaryotic cell is a cultured yeast cell.

17. The method of claim 15 wherein said protein includes at least one amino acid substitution of a cysteine residue.

18. 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 DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.

19. The method of claim 18 wherein said eucaryotic host cell is a yeast cell.

20. The method of claim 18 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-109.

21. The method of claim 18 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-97 fused inreading frame to A-chain amino acids 92-104.

22. The method of claim 18 wherein said protein includes at least one amino acid substitution of a cysteine residue.

23. 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 DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.

24. The method of claim 23 wherein said cultured eucaryotic cell is a cultured cell.

25. The method of claim 23 wherein said protein is a monomeric species having biological activity.

26. The method of claim 23 wherein said protein includes at least one amino acid substitution of a cysteine

27. 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 transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.

28. A eucaryotic host cell transformed with a vector capable of directing the expression and secretion biologically active PDGF analogs in eucaryotic cells, said vector containing a transcriptional promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the A-chain of PDGF.

29. The eucaryotic host cell of claim 28 wherein said cultured eucaryotic cell is a cultured yeast cell.

30. The eucaryotic host cell of claim 28 wherein said protein includes at least one amino acid substitution of a cysteine residue.

31. A eucaryotic host 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 promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to the B-chain of PDGF.

32. The eucaryotic host cell of claim 29 wherein said protein contains a proteolytic processing site, and said vector further encodes a prepropolypeptide lacking a proteolytic processing site, thereby resulting in proteolytic processing with the B-chain.

33. The eucaryotic host cell of claim 29 wherein said protein lacks substantially the first twenty-eight amino acids of the B-chain of PDGF.

34. The eucaryotic host cell of claim 29 wherein said protein is a monomeric species having biological activity.

35. The eucaryotic host cell of claim 29 wherein said protein includes at least one amino acid substitution of a cysteine residue.

36. A eucaryotic host 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 promoter followed downstream by a DNA sequence, a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the A-chain of PDGF, and a portion of said DNA sequence encoding a protein which is substantially homologous to at least a portion of the B-chain of PDGF, said portions of said DNA sequence encoding a protein having substantially the same biological activity as PDGF.

37. The eucaryotic host cell of claim 34 wherein said cultured eucaryotic cell is a cultured yeast cell.

38. The eucaryotic host cell of claim 34 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-109.

39. The eucaryotic host cell of claim 34 wherein said DNA sequence encodes a protein substantially homologous to A-chain amino acids 1-17 fused in reading frame to B-chain amino acids 24-97 fuse in reading frame to A-chain amino acids 92-104.

40. The eucaryotic host cell of claim 34 wherein said protein includes at least one amino acid substitution of a cysteine residue.

41. A eucaryotic host cell transformed with a vector capable of directing the expression and secretion of biologically active PDGF analogs in eucaryotic cells, said vector containing transcriptional promoters followed downstream by DNA sequences encoding polypeptide chains substantially homologous to the A- and B-chains of PDGF, said chains forming a heterodimer.

42. The eucaryotic host cell of claim 39 wherein said cultured eucaryotic cell is a cultured yeast cell.

43. A eucaryotic host 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 promoter followed downstream by a DNA sequence encoding a prepropolypetide lacking a proteolytic processing site, said DNA sequence being followed downstream by a DNA sequence encoding a protein containing a proteolytic processing site which is substantially homologous to the B-chain of PDGF thereby resulting in proteolytic processing within the B-chain of PDGF.

44. A eucaryotic host 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 promoter followed downstream by a DNA sequence encoding a protein which is substantially homologous to a portion of the B-chain of PDGF from amino acid 29 to amino acid 109.
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