Expression of procollagen in yeast

• US 6,472,171 B1
• Filed: 07/22/1998
• Issued: 10/29/2002
• Est. Priority Date: 10/20/1995
• Status: Expired due to Term

• Current Assignee: Genotypes, Inc., Surgical Specialties Corporation Limited (Ssc Holdco Ltd)
• Original Assignee: Cohesion Technologies Inc. (Angiotech Pharmaceuticals Incorporated), Genotypes, Inc.
• Inventors: Chisholm, George E., Toman, David P., Daniels, Gregory A., Berg, Richard A., Hitzeman, Ronald A.
• Primary Examiner(s): Carlson, Karen Cochrane
• Assistant Examiner(s): Robinson, Hope A.
• Application Number:
  US09/120,561
• Time in Patent Office: 1,560 Days
• Field of Search: 435/69.1, 435/69.8, 435/233, 435/252.3, 435/254.1, 435/320.1, 530/350, 530/356, 536/23.1, 536/23.5, 536/24.1, 536/23.2
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/90 Isomerases (5.)

Abstract

The invention discloses methods for effecting the production of recombinant mammalian procollagen in yeast, as well as compositions comprising yeast cells cap producing mammalian procollagen.

Claims

1. A method for making a mammalian collagen or procollagen in yeast, the method comprising the steps of:

(a) incubating a yeast cell comprising;

(i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter;

(ii) a prolyl hydroxylase gene;

(iii) a protein disulfide isomerase gene, wherein the prolyl hydroxylase and protein disulfide isomerase genes are expressed to produce polypeptides that are associated into active proteins during the incubating step, in a medium under conditions wherein the collagen genes are expressed as procollagen chains and the chains secreted in the form of triple helical procollagen; and

(b) recovering the triple helical procollagen.

2. The method of claim 1, wherein the first single divergent heterologous promoter comprises a GAL1-10 promoter.

3. The method of claim 1, wherein the collagen genes reside on a plasmid or multiple integrants in the cell.

4. The method of claim 1, wherein the prolyl hydroxylase and protein disulfide isomerase genes are integrated into the genome of the yeast.

5. The method of claim 1, wherein the prolyl hydroxylase gene and protein disulfide isomerase genes are operably linked to a second single divergent heterologous promoter.

6. The method of claim 1, wherein the first and second single divergent heterologous promoters consist essentially of the same nucleotide sequence.

7. The method of claim 1, wherein the protein disulfide isomerase gene comprises a heterologous signal sequence.

8. The method of claim 1, wherein the medium comprises one or more supplemental amino acids which are degraded in the cell to α

-ketoglutarate sufficient to promote the hydroxylation of the collagen under the conditions.

9. The method of claim 1, wherein the yeast cell is a Saccharomyces cerevisiae.

10. The method of claim 1, wherein the mammalian collagen genes are human collagen genes.

11. A method according to claim 1 wherein the procollagen is heterotrimeric.

12. A method according to claim 1 wherein the collagen genes encode different collagen chains.

13. A method for making a mammalian collagen or procollagen in yeast, the method comprising the steps of:

(a) incubating a yeast cell comprising;

(i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter;

(ii) a prolyl hydroxylase gene;

(iii) a protein disulfide isomerase gene, wherein the prolyl hydroxylase and protein disulfide isomerase genes are expressed to produce polypeptides that are associated into active proteins during the incubating step, in a medium under conditions wherein the collagen genes are expressed as procollagen chains and the chains secreted in the form of triple helical procollagen,

wherein the yeast cell is Saccharomyces cerevisiae, the prolyl hydroxylase and protein disulfide isomerase genes are integrated into the genome of the yeast, the first single divergent heterologous promoter is a GAL1-10 promoter, the collagen genes reside on a plasmid in the yeast, the prolyl hydroxylase gene and protein disulfide isomerase genes are operably linked to a second single divergent heterologous promoter, the first and second single divergent heterologous promoters are the same, and the mammalian collagen genes are human collagen genes; and

(b) recovering the triple helical procollagen.

14. A method according to claim 13 wherein the procollagen is heterotrimeric.

15. A yeast cell comprising:

(i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter;

(ii) a prolyl hydroxylase gene; and

(iii) a protein disulfide isomerase gene.

16. The cell of claim 15, wherein the first single divergent heterologous promoter comprises a GAL1-10 promoter.

17. The cell of claim 15, wherein the collagen genes reside on a plasmid or multiple integrants in the cell.

18. The cell of claim 15, wherein the prolyl hydroxylase and protein disulfide isomerase genes are integrated into the genome of the yeast.

19. The cell of claim 15, wherein the prolyl hydroxylase gene and protein disulfide isomerase genes are operably linked to a second single divergent heterologous promoter.

20. The cell of claim 15, wherein the first and second single divergent heterologous promoters consist essentially of the same nucleotide sequence.

21. The cell of claim 15, wherein the protein disulfide isomerase gene comprises a heterologous signal sequence.

22. The cell of claim 15, wherein the yeast cell is a Saccharomyces cerevisiae.

23. The cell of claim 15, wherein the mammalian collagen genes are human collagen genes.

24. The cell of claim 15, wherein the first single divergent heterologous promoter is a GAL1-10 promoter, the collagen genes reside on a plasmid in the cell, the prolyl hydroxylase and protein disulfide isomerase genes are integrated into the genome of the yeast, the prolyl hydroxylase gene and protein disulfide isomerase genes are operably linked to a second single divergent heterologous promoter, the first and second single divergent heterologous promoters are the same, the protein disulfide isomerase gene comprises a signal sequence heterologous to the protein disulfide isomerase gene, the yeast cell is a Saccharomyces cerevisiae, the mammalian collagen genes are human collagen genes.

25. A method according to claim 24 wherein the collagen genes encode different collagen chains.