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Claims for Patent: 6,103,515

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Claims for Patent: 6,103,515

Title: Production of polypeptides by use of novel protease deficient yeast strains
Abstract:A novel process for the production of heterologous proteins including the use of certain transformed protease deficient yeast strains is provided. The invention concerns also said transformed yeast strains and methods for the production thereof.
Inventor(s): Treichler; Hansjorg (Kanerkinden, CH), Takabayashi; Kenji (Basel, CH), Wolf; Dieter Heinrich (Gundelfingen, DE), Heim; Jutta (Ramlinsburg, CH)
Assignee: Novartis Corporation (New York, NY) UCP Gen-Pharma AG (Kirchberg, CH)
Application Number:07/895,581
Patent Claims: 1. A Saccharomyces cerevisiae strain which lacks carboxypeptidase ysc.alpha. activity and lacks proteolytic activity selected from the group consisting of proteolytic yscA, yscB, yscY and yscS activity and has been transformed with a hybrid vector comprising a Saccharomyces cerevisiae promoter operably linked to a DNA coding for a full-length mature protein which bears no basic C-terminal amino acids and which is selected from the group consisting of hANP, EGF, connective tissue activating peptide-III and desulphatohirudin.

2. A method for the production of a Saccharomyces cerevisiae strain which lacks carboxypeptidase ysc.alpha. activity and lacks proteolytic activity selected from the group consisting of proteolytic yscA, yscB, yscY and yscS activity and has been transformed with a hybrid vector comprising a Saccharomyces cerevisiae promoter operably linked to a DNA coding for a full-length mature protein which bears no basic C-terminal amino acids and which is selected from the group consisting of hANP, EGF, connective tissue activating peptide-III and desulphatohirudin, comprising transforming a Saccharomyces cerevisiae strain which lacks carboxypeptidase ysc.alpha. activity and lacks proteolytic activity selected from the group consisting of proteolytic yscA, yscb, yscY and yscS activity with said hybrid vector.

3. A method for the production of a full-length mature protein which bears no basic C-terminal amino acids and which is selected from the group consisting of hANP, EGF, connective tissue activating peptide-III and desulphatohirudin, comprising culturing a Saccharomyces cerevisiae strain which lacks carboxypeptidase ysc.alpha. activity and lacks proteolytic activity selected from the group consisting of proteolytic yscA, yscB, yscY and yscS activity and has been transformed with a hybrid vector comprising a Saccharomyces cerevisiae promoter operably linked to a DNA coding for said protein, and isolating said protein.

4. A Saccharomyces cerevisiae strain according to claim 1, wherein said hybrid vector comprises a Saccharomyces cerevisiae promoter operably linked to a first DNA encoding a signal peptide linked in a proper reading frame to a second DNA encoding said full-length mature protein and a DNA containing Saccharomyces cerevisiae transcription termination signals.

5. A Saccharomyces cerevisiae strain according to claim 1, wherein said full-length mature protein is desulphatohirudin.

6. A Saccharomyces cerevisiae strain according to claim 1, which is free of endogenous two-micron plasmid and has been transformed with a hybrid vector comprising the complete two-micron DNA comprising intact REP1, REP2 and FLP genes and intact ORI, STB, IR1 AND IR2 sites.

7. A Saccharomyces cerevisiae strain according to claim 1, wherein said hybrid vector comprises a Saccharomyces cerevisiae promoter selected from the group consisting of the MF.alpha.1 promoter, GAL1 promoter, a promoter of a gene encoding a glycolytic enzyme, ADHI promoter, TRPI promoter, PHO5 and the PHO5 promoter in which the upstream activation sites have been deleted.

8. A Saccharomyces cerevisiae strain according to claim 4, wherein said hybrid vector comprises a first DNA selected from the group consisting of the hirudin signal sequence, the signal and prepro sequences of the yeast invertase, .alpha.-factor pheromone peptidase (KEX1), "Killer toxin" and repressible acid phosphatase (PHO5) genes, and the glucoamylase signal sequence from Aspergillus awamori.

9. A method for the production of an essentially homogeneous mature protein according to claim 3, wherein said hybrid vector comprises a Saccharomyces cerevisiae promoter operably linked to a first DNA encoding a signal peptide linked in the proper reading frame to a second DNA coding for said protein and a DNA sequence containing Saccharomyces cerevisiae transcription termination signals.

10. A method for the production of an essentially homogenous mature protein

according to claim 9, wherein said protein is desulphatohirudin.

11. A method according to claim 10 for the preparation of desulphatohirudin variant HV1.
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