Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
View Patent ↗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.
1. A method for the synthesis of a desired secreted heteromultimeric protein comprising at least two non-identical subunit polypeptide chains in a diploid Pichia yeast cell, the method comprising: (i) producing a recombinant diploid Pichia yeast by spheroplast fusion of 2 haploid Pichia spheroplasts or by mating 2 intact haploid Pichia yeast cells, resulting in a diploid Pichia yeast comprising nucleic acids encoding all of the subunits of a desired heterologous (non-yeast)-heteromultimeric, protein, which nucleic acids are each operably linked to a promoter and signal peptide that provides for the expression and secretion of the subunits encoded by the nucleic acids by said recombinant diploid Pichia yeast; and (ii) culturing said recombinant diploid Pichia yeast under conditions that provide for the expression and secretion of the desired heteromultimeric protein into a culture medium comprising the recombinant diploid Pichia yeast.
2. The method of claim 1 for the synthesis of a secreted heteromultimeric protein comprising at least two non-identical subunit polypeptide chains in a diploid Pichia yeast cell, the method comprising: transforming a first intact haploid Pichia cell with a first expression vector, said expression vector comprising a nucleic acid encoding at least a first subunit of said protein, operably linked to a first yeast promoter and signal sequence; transforming a second intact Pichia haploid cell with a second expression vector, said expression vector comprising nucleic acids encoding the remaining subunits of said desired heteromultimeric protein, said nucleic acids each being operably linked to a second promoter and signal sequence; generating a diploid cell from said first and second Pichia intact yeast haploid cells by mating; and culturing said diploid cell under conditions wherein the subunits comprising the desired heteromultimeric protein are expressed and secreted as the desired multimeric protein.
3. The method according to claim 1 , wherein said Pichia species is selected from the group consisting of Pichia pastoris, Pichia methanolica , and Pichia angusta.
4. The method according to claim 1 , wherein said heteromultimeric protein is a mammalian protein.
5. The method according to claim 1 , wherein said heteromultimeric protein is an antibody comprising at least a variable region of a heavy and a light chain.
6. The method according to claim 5 , wherein said heteromultimeric protein is an intact antibody.
7. The method according to claim 1 , wherein said diploidal Pichia yeast is obtained by mating 2 intact haploid Pichia cells or fusing 2 haploid Pichia spheroplasts, wherein said 2 intact haploid Pichia cells or spheroplasts respectively comprise complementary Pichia auxotrophs.
8. The method according to claim 1 , further comprising the step of calibrating the level of expression of said subunits prior to generating said diploid P Pichia yeast by fusion of 2 haploid Pichia spheroplasts or mating of 2 intact haploid Pichia yeast cells.
9. The method according to claim 2 , wherein all of the promoters which are operably linked to the nucleic acids encoding the subunits are the same.
10. The method according to claim 2 , wherein all of the promoters operably linked to the nucleic acids encoding the subunits are not identical.
11. The method according to claim 2 , wherein said first promoter and said second promoter are different.
12. The method according to claim 1 , wherein said promoters are constitutive promoters.
13. The method according to claim 1 , wherein said promoters are inducible promoters.
14. The method according to claim 1 , wherein at least one of said promoters is a GAP promoter.
15. The method according to claim 1 , wherein said culturing step is performed in minimal media.
16. The method according to claim 15 , wherein said minimal media lacks selective agents.
17. The method according to claim 1 , wherein said multimeric protein is secreted by said diploid cells at a concentration of at least about 100 mg/liter culture.
18. The method of claim 1 , for the synthesis of a desired secreted heteromultimeric protein comprising at least two non-identical subunit polypeptide chains in a diploid Pichia yeast cell, the method comprising: (i) producing a recombinant diploid Pichia yeast by spheroplast fusion of 2 haploid Pichia spheroplast Pichia yeast cells, resulting in a diploid Pichia yeast comprising nucleic acids encoding all of the subunits of a desired heterologous (non-yeast) heteromultimeric protein, which nucleic acids are each operably linked to a promoter and signal peptide that provides for the expression and secretion of the subunits encoded by the nucleic acids by said recombinant diploid Pichia yeast; and (ii) culturing said recombinant diploid Pichia yeast under conditions that provide for the expression and secretion of the desired heteromultimeric protein into a culture medium comprising the recombinant diploid Pichia yeast.
19. The method according to claim 18 , wherein all of the promoters operably linked to the nucleic acids encoding the subunits are not identical.
20. The method according to claim 18 , wherein said first promoter and said second promoter are different.
21. The method according to claim 1 , wherein said Pichia species is Pichia pastoris.
22. The method according to claim 5 , wherein said Pichia species is Pichia pastoris.
23. The method according to claim 6 , wherein said Pichia species is Pichia pastoris.