Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy
AssigneesKeck Graduate Institute, Alder Biopharmaceuticals, Inc.
InventorsJames M. Cregg, John Latham, Mark Litton, Randall Schatzman, Ilya I. Tolstorukov
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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 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.
Dependencies are inferred from the claim text.