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 signal 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 wherein said precursor of human insulin 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 the structure:
- 5'-AF-CHO-X.sub.n -S-Gene*-3'
- wherein
- AF encodes a yeast alpha-factor signal peptide;
- CHO encodes a yeast alpha-factor precursor glycosylation site;
- 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 no 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.
Parent Case Info
This application is a continuation of application Ser. No. 07/864,206, filed 3 Apr. 1992, now abandoned, which is a continuation of application Ser. No. 07/670,352, filed 13 Mar. 1991, now abandoned, which is a continuation of application Ser. No. 07/530,477, filed 29 May 1990, now abandoned, which is a continuation of application Ser. No. 07/139,682, filed 30 Dec. 1987, now abandoned.
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4546082 |
Kurjan et al. |
Oct 1985 |
|
4870008 |
Brake |
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|
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Goodey et al. |
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Foreign Referenced Citations (2)
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Continuations (4)
|
Number |
Date |
Country |
Parent |
864206 |
Apr 1992 |
|
Parent |
670352 |
Mar 1991 |
|
Parent |
530477 |
May 1990 |
|
Parent |
139682 |
Dec 1987 |
|