Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
AssigneesKeck Graduate Institute, H. Lundbeck A/S
InventorsJames M. Cregg, John A. Latham, Mark Litton, Randall Schatzman, Ilya I. Tolstrorukov
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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.
Claims23
A method for the synthesis of a secreted heteromultimeric protein comprising at least two non-identical subunit polypeptide chains in a Pichia pastoris diploid cell, the method comprising:
- (a) transforming a first auxotrophic Pichia pastoris haploid cell with a linearized first expression vector, said expression vector comprising a nucleic acid sequence encoding a first subunit of said heteromultimeric protein, operably linked to a first Pichia pastoris promoter whereby the first expression vector comprising a nucleic acid sequence encoding the first subunit of said protein becomes integrated into the gene locus comprising said Pichia pastoris promoter;
- (b) transforming a second auxotrophic Pichia pastoris haploid cell with a linearized second expression vector, said expression vector comprising a nucleic acid sequence encoding a second subunit of said heteromultimeric protein, operably linked to a second Pichia pastoris promoter whereby the second expression vector comprising said nucleic acid sequence encoding said second subunit of said heteromultimeric protein becomes integrated into the gene locus comprising said second Pichia pastoris promoter;
- (c) generating a putative diploid Pichia pastoris cell by mating or fusion of said first and second Pichia pastoris haploid cells;
- (d) determining whether the putative diploid cell is diploid by (i) permitting said putative diploid cell to go through meiosis and form spores;
- (ii) determining if a significant percentage of the resulting spore products are single or double auxotrophs and (iii) based thereon determining that the putative diploid Pichia pastoris cell is diploid;
- and
- (e) culturing said diploid Pichia pastoris cell in a culture medium under conditions wherein said first and said second subunits are expressed by said diploid Pichia pastoris cell and secreted as said heteromultimeric protein;
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- (f) isolating the secreted heteromultimeric protein from the culture medium.
Dependencies are inferred from the claim text.