Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1.
The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1beta, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.
Lodi
Tiziana
T
Department of Genetics, Anthropology, and Evolution, University of Parma, Parco Area delle Scienze 11/A, I-43100 Parma, Italy.
Neglia
Barbara
B
Donnini
Claudia
C
D004721:Endoplasmic Reticulum; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D020440:Gene Duplication; D015966:Gene Expression Regulation, Fungal; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007716:Kluyveromyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012709:Serum Albumin / Q000378:metabolism*
D005656:Fungal Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012709:Serum Albumin; D050862:Oxidoreductases Acting on Sulfur Group Donors; C113186:ERO1 protein, S cerevisiae; D019704:Protein Disulfide-Isomerases