Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

Patent metadata

Field	Value
Publication number	US-10155819-B2
Application number	US-201414165896-A
Country	US
Kind code	B2
Filing date	Jan 28, 2014
Priority date	Oct 22, 2003
Publication date	Dec 18, 2018
Grant date	Dec 18, 2018

Abstract

Methods are provided for the synthesis and secretion of recombinant hetero-multimeric proteins in mating competent yeast. A first expression vector is transformed into a first haploid cell; and a second expression vector is transformed into a second haploid cell. The transformed haploid cells, each individually synthesizing a non-identical polypeptide, are identified and then genetically crossed or fused. The resulting diploid strains are utilized to produce and secrete fully assembled and biologically functional hetero-multimeric protein.

First claim

What is claimed is:

1. A method for producing a diploid Pichia yeast strain capable of expression and secretion of a desired heterologous heteromultimeric protein into a culture medium containing said diploid Pichia yeast strain; wherein said heteromultimeric protein comprises at least two non-identical subunit polypeptide chains, the method comprising:
(i) obtaining a first haploid Pichia yeast strain which comprises a nucleic acid sequence encoding at least one subunit of a heterologous heteromultimeric protein, which coding sequence is operably linked to a promoter and a signal sequence that directs the secretion of said subunit by a Pichia yeast;
(ii) obtaining a second haploid Pichia yeast which comprises a nucleic acid sequence encoding at least one subunit of the same heterologous heteromultimeric protein, wherein the subunits encoded by the first and second haploid Pichia yeast strains comprise all of the subunits of the desired heterologous heteromultimeric protein, and which coding sequence is operably linked to a promoter and a signal sequence that directs the secretion of said subunit by a Pichia yeast;
(iii) fusing said first and second haploid Pichia yeast strains by spheroplast fusion or by mating to produce a diploid Pichia yeast strain which is capable of expression and secretion of the desired heterologous heteromultimeric protein into a culture medium, wherein said first and second haploid Pichia yeast strains are of the same species;
(iv) culturing the resultant diploid Pichia yeast strain under culture conditions wherein at least about 50 mg/L of said heterologous heteromultimeric protein is secreted into the culture medium during said culture period; and
(v) recovering the secreted heterologous heteromultimeric protein from the culture medium.

2. The method of claim 1 , wherein the diploid Pichia yeast strain is produced by mating.

3. The method of claim 1 , wherein the diploid Pichia yeast strain is produced by spheroplast fusion.

4. The method according to claim 1 , wherein both said first and second haploid Pichia yeast strains are Pichia pastoris, Pichia methanolica, or Pichia angusta.

5. The method according to claim 2 , wherein both said first and second haploid Pichia yeast strains are Pichia pastoris, Pichia methanolica, or Pichia angusta.

6. The method according to claim 3 , wherein both said first and second haploid Pichia yeast strains are Pichia pastoris, Pichia methanolica, or Pichia angusta.

7. The method according to claim 1 , wherein said heterologous heteromultimeric protein is a mammalian protein.

8. The method according to claim 1 , wherein said heterologous heteromultimeric protein is an intact antibody comprising heavy and light variable regions and human constant regions.

9. The method according to claim 1 , wherein said heterologous heteromultimeric protein is a human polypeptide.

10. The method according to claim 1 , wherein both said first and said second haploid Pichia yeast strains are auxotrophic.

11. The method according to claim 1 , wherein in steps (i) and (ii) the nucleic acid sequences encoding the subunits are respectively operably linked to a first promoter and a second promoter which first and second promoters are the same or different.

12. The method according to claim 11 , wherein said first and second promoters are both inducible promoters.

13. The method according to claim 11 , wherein at least one of said first and second promoters is a GAP promoter.

14. The method of claim 1 , wherein both said first and said second haploid Pichia yeast strains are Pichia pastoris strains.

15. The method of claim 2 , wherein both said first and said second haploid Pichia yeast strains are Pichia pastoris strains.

16. The method of claim 3 , wherein both said first and said second haploid Pichia yeast strains are Pichia pastoris strains.

17. The method of claim 8 , wherein both said first and said second haploid Pichia yeast strains are Pichia pastoris strains.

18. The method of claim 9 , wherein both said first and said second haploid Pichia yeast strains are Pichia pastoris strains.

19. The method of claim 1 , wherein the culture period comprises culturing the diploid Pichia cells for at least 20 doublings and after said at least 20 doublings continue to express said heterologous heteromultimeric protein.

20. The method of claim 19 , wherein the culture period comprises culturing the diploid Pichia cells for at least 50 doublings and after said at least 50 doublings continue to express said heterologous heteromultimeric protein.

21. The method of claim 19 , wherein the culture period comprises culturing the diploid Pichia cells for at least 100 doublings and after said at least 100 doublings continue to express said heterologous heteromultimeric protein.

Assignees

• Alderbio Holdings Llc
• Keck Graduate Inst

Inventors

• Cregg James M
• Latham John
• Litton Mark
• Schatzman Randal
• Tolstrorukov Ilya

Classifications

• C12N15/81

  for yeasts · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K16/2896 Primary

  against molecules with a "CD"-designation, not provided for elsewhere · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title

• C07K16/18

  against material from animals or humans · CPC title