Claims for Patent: RE37343

Summary for Patent: RE37343

Title:	Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences
Abstract:	A yeast .alpha.-factor expression system is provided comprised of a truncated leader sequence, containing the .alpha.-factor signal peptide and one glycosylation site, linked by a processing site to a non-yeast protein-encoding sequence.
Inventor(s):	Tekamp-Olson; Patricia (San Anselmo, CA)
Assignee:	Chiron Corporation (Emeryville, CA)
Application Number:	09/140,858
Patent Claims:	1. A yeast cell comprising a DNA construct that provides for the expression and secretion of a non-yeast protein, said DNA construct comprising a yeast recognized transcription initiation sequence, linked 5' to a coding sequence under the control of both said yeast recognized transcription initiation sequence and a yeast-recognized termination sequence, said yeast-recognized termination sequence being 3' to said coding sequence, wherein said coding sequence encodes a precursor polypeptide comprised of a leader sequence and said non-yeast protein linked by a processing site that provides for the cleavage of said non-yeast protein from said precursor polypeptide, wherein said leader sequence is about the first 25 to about the first 50 N-terminal residues of a yeast alpha-factor leader polypeptide, comprises a single yeast alpha-factor precursor glycosylation site and comprises a single peptide of a yeast alpha-factor precursor comprising the first about 19 to about 23 N-terminal residues of said alpha-factor precursor. 2. The cell of claim 1 wherein said non-yeast protein is a mammalian protein. 3. The cell of claim 2 wherein said mammalian protein is a precursor of human insulin. 4. The cell of claim 3 wherein said precursor of human insulin is human proinsulin. 5. The cell of claim .[.3.]. .Iadd.1 .Iaddend.wherein said .[.precursor of human insulin.]. .Iadd.non-yeast protein .Iaddend.comprises insulin a chain and insulin b chain linked by a yeast-recognized processing site cleaved in vivo. 6. The cell of claim 5 wherein said processing site is cleaved by the KEX2 gene product of Saccharomyces. 7. The cell of claim 2 wherein said mammalian protein is insulin-like growth factor I. 8. The cell of claim 1 wherein said yeast cell is from the genus Saccharomyces. 9. The cell of claim 8 wherein said yeast cell is S. cerevisiae. 10. The cell of claim 8 wherein yeast .alpha.-factor precursor is S. cerevisiae MF.alpha.1. 11. The cell of claim 1 wherein said leader sequence is about 28 to about 40 N-terminal residues of said precursor polypeptide. 12. The cell of claim 1 wherein said leader sequence is about 35 contiguous N-terminal residues of a yeast alpha-factor precursor polypeptide. 13. A double-stranded DNA molecule comprising a region encoding a precursor polypeptide secretable by a yeast host, said region, with reference to one of the strands, .[.having.]. .Iadd.comprising .Iaddend.the structure: wherein AF encodes a yeast alpha-factor signal peptide; CHO encodes a .[.yeast alpha-factor precursor.]. glycosylation site .Iadd.comprising the amino acid sequence Asn-Y-Y' wherein Y is any amino acid and Y' is Thr or Ser;.Iaddend. X.sub.n encodes a spacer polypeptide of n amino acids in length that does not contain a glycosylation site or a processing site that provides for cleavage of said precursor polypeptide in vivo by yeast; n is an integer from 0 to 30; Gene* encodes a non-yeast protein; and S encodes a processing site that provides for cleavage of said precursor polypeptide. 14. The DNA molecule of claim 13 wherein AF encodes a polypeptide of about 19 to 23 amino acids in length. 15. The DNA molecule of claim 13 wherein n is an integer from about 0 to about 20. 16. The DNA molecule of claim 13 wherein n is an integer from about 0 to about 10. 17. The DNA molecule of claim 13 wherein n is an integer from about 3 to about 10. 18. The DNA molecule of claim 13 wherein said yeast host is a Saccharomyces. 19. The DNA molecule of claim 13 wherein said yeast .alpha.-factor signal peptide is a Saccharomyces signal peptide. 20. The DNA molecule of claim 13 wherein S encodes a processing site recognized in vivo by said yeast host. 21. The DNA molecule of claim 20 wherein S encodes a dipeptide recognized by the KEX2 endopeptidase. 22. The DNA molecule of claim 21 wherein said dipeptide is 5'-Lys-Arg-3' or 5'-Arg-Arg-3'. 23. The DNA molecule of claim 13 comprising a replicon. 24. The DNA molecule of claim 23 wherein said region encoding said precursor polypeptide is under the control of yeast-recognized transcription initiation and termination sequences, and said replicon is a yeast replicon. 25. The DNA molecule of claim 24 wherein said replicon is a plasmid. 26. The DNA molecule of claim 24 wherein said replicon is a chromosome. 27. A DNA molecule comprising a nucleotide sequence that encodes about the first 25 to about the first 50 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site. 28. The DNA molecule of claim 27 wherein the nucleotide sequence encodes about the first 25 to about the first 40 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site. 29. The DNA molecule of claim 27 wherein the nucleotide sequence encodes about the first 35 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and not other yeast alpha-factor precursor leader sequence glycosylation site. 30. The DNA molecule of claim 27 wherein the nucleotide sequence encodes about the first 28 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site. 31. The DNA molecule of claim 27 wherein the nucleotide sequence encodes amino acids 1-25 and 81-83 of a yeast alpha-factor precursor leader sequence..Iadd. 32. A method for producing a recombinant protein, said method comprising: providing a yeast cell as defined in claim 1, and culturing said yeast cell under conditions that provide for expression and secretion of said non-yeast protein..Iaddend..Iadd. 33. The method of claim 32 wherein said non-yeast protein is a mammalian protein..Iaddend..Iadd. 34. The method of claim 32 wherein said non-yeast protein is a precursor of human insulin..Iaddend..Iadd. 35. The method of claim 34 wherein said precursor of human insulin is human proinsulin..Iaddend..Iadd. 36. The method of claim 34 wherein said precursor of human insulin comprises insulin a chain and insulin b chain linked by a yeast-recognized processing site cleaved in vivo..Iaddend..Iadd. 37. The method of claim 36 wherein said processing site is cleaved by the KEX2 gene product of Saccharomyces..Iaddend..Iadd. 38. The method of claim 33 wherein said mammalian protein is insulin-like growth factor I..Iaddend..Iadd. 39. The method of claim 32 wherein said yeast cell is from the genus Saccharomyces..Iaddend..Iadd. 40. The method of claim 39 wherein said yeast cell is S. cerevisiae..Iaddend..Iadd. 41. The method of claim 39 wherein said yeast alpha-factor leader polypeptide is from S. cerevisiae MF.alpha.1..Iaddend..Iadd. 42. The method of claim 32 wherein said leader sequence is about the first 28 to about the first 40 N-terminal amino acid residues of a yeast alpha-factor leader polypeptide..Iaddend..Iadd. 43. The method of claim 32 wherein said leader sequence is about the first 35 contiguous N-terminal residues of a yeast alpha-factor leader polypeptide..Iaddend..Iadd. 44. A method of producing a recombinant protein, said method comprising: transforming a yeast cell with a DNA construct that provides for the expression and secretion of a non-yeast protein, wherein said DNA construct comprises the double-stranded DNA molecule as defined in claim 13, and culturing said transformed yeast cell under conditions that provide for expression and secretion of said non-yeast protein..Iaddend..Iadd. 45. The method of claim 44 wherein AF encodes a polypeptide of about 19 to 23 amino acids in length..Iaddend..Iadd. 46. The method of claim 44 wherein n is an integer from about 0 to about 20..Iaddend..Iadd. 47. The method of claim 44 wherein n is an integer from about 0 to about 10..Iaddend..Iadd. 48. The method of claim 44 wherein n is an integer from about 3 to about 10..Iaddend..Iadd. 49. The method of claim 44 wherein said yeast host is a Saccharomyces..Iaddend..Iadd. 50. The method of claim 44 wherein said yeast alpha-factor signal peptide is a Saccharomyces signal peptide..Iaddend..Iadd. 51. The method of claim 44 wherein S encodes a processing site recognized in vivo by said yeast host..Iaddend..Iadd. 52. The method of claim 51 wherein S encodes a dipeptide recognized by the KEX2 endopeptidase..Iaddend..Iadd. 53. The method of claim 52 wherein said dipeptide is 5'-Lys-Arg-3' or 5'-Arg-Arg-3'..Iaddend..Iadd. 54. The method of claim 44 wherein said DNA molecule comprises a replicon..Iaddend..Iadd. 55. The method of claim 54 wherein said region encoding said precursor polypeptide is under the control of yeast-recognized transcription initiation and termination sequences, and said replicon is a yeast replicon..Iaddend..Iadd. 56. The method of claim 55 wherein said replicon is a plasmid..Iaddend..Iadd. 57. The method of claim 55 wherein said replicon is a chromosome..Iaddend..Iadd. 58. A method of producing a recombinant protein, said method comprising: transforming a yeast cell with a DNA construct that provides for the expression and secretion of a non-yeast protein, wherein said DNA construct comprises the double-stranded DNA molecule as defined in claim 27, and culturing said transformed yeast cell under conditions that provide for expression and secretion of said non-yeast protein..Iaddend..Iadd. 59. The method of claim 58 wherein the nucleotide sequence encodes about the first 25 to about the first 40 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site..Iaddend..Iadd. 60. The method of claim 58 wherein the nucleotide sequence encodes about the first 35 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site..Iaddend..Iadd. 61. The method of claim 58 wherein the nucleotide sequence encodes about the first 28 contiguous N-terminal amino acids of a yeast alpha-factor precursor leader sequence that includes a single yeast alpha-factor precursor leader sequence glycosylation site and no other yeast alpha-factor precursor leader sequence glycosylation site..Iaddend..Iadd. 62. The method of claim 59 wherein the nucleotide sequence encodes amino acids 1-25 linked to amino acids 81-83 of a yeast alpha-factor precursor leader sequence..Iaddend..Iadd. 63. The method of claim 44 wherein CHO encodes a yeast alpha-factor precursor glycosylation site..Iaddend..Iadd. 64. The DNA molecule of claim 13 wherein CHO encodes a yeast alpha-factor precursor glycosylation site..Iaddend..Iadd. 65. The method of claim 49 wherein said yeast host is S. cerevisiae..Iaddend..Iadd. 66. The method of claim 49 wherein the leader construct AF-CHO-X.sub.n -S is about the first 25 to about the first 40 contiguous N-terminal residues of a yeast alpha-factor signal peptide..Iaddend..Iadd. 67. The method of claim 49 wherein the leader construct AF-CHO-X.sub.n -S is about the first 28 to about the first 40 contiguous N-terminal residues of a yeast alpha-factor signal peptide..Iaddend..Iadd. 68. The method of claim 49 wherein the leader construct AF-CHO-X.sub.n -S is about the first 35 to about the first 40 contiguous N-terminal residues of yeast alpha-factor signal peptide..Iaddend..Iadd. 69. The method of claim 68 wherein said non-yeast protein is a human proinsulin..Iaddend..Iadd. 70. The method of claim 69 wherein said DNA construct is plasmid pYGAI7 (ATCC Accession Number 67597)..Iaddend..Iadd. 71. The method of claim 69 wherein said human proinsulin comprises insulin a chain and insulin b chain linked by a yeast-recognized processing site cleaved in vivo by the KEX2 gene product of Saccharomyces..Iaddend..Iadd. 72. The method of claim 71 wherein said DNA construct is plasmid pY.alpha.f.sub.L 7C3(ATCC Accession Number 67596)..Iaddend..Iadd. 73. The method of claim 49 wherein the leader construct AF-CHO-X.sub.n -S is amino acids 1-25 linked to amino acids 81-83 of a yeast alpha-factor signal peptide..Iaddend..Iadd. 74. The method of claim 73 wherein said non-yeast protein is insulin-like growth factor I..Iaddend..Iadd. 75. The method of claim 74 wherein said DNA construct is plasmid pYLUIGFI-55(ATCC Accession Number 67595)..Iaddend.

Details for Patent RE37343

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	10/28/1982		2040-03-31
Eli Lilly And Company	HUMULIN R U-500	insulin human	Injection	018780	12/29/2015		2040-03-31
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	08/06/1998		2040-03-31
Eli Lilly And Company	HUMULIN R U-500	insulin human	Injection	018780	03/31/1994		2040-03-31
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	05/25/2018		2040-03-31
Baxter Healthcare Corporation	MYXREDLIN	insulin human	Injection	208157	06/20/2019		2040-03-31