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Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon‐α2b fusion protein
Author(s) -
Zhao Hong Liang,
Xue Chong,
Wang Yang,
Duan Qing Fen,
Xiong Xiang Hua,
Yao Xue Qin,
Liu Zhi Min
Publication year - 2008
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1589
Subject(s) - pichia pastoris , heterologous , biology , human serum albumin , mutagenesis , secretion , mutant , fusion protein , viral matrix protein , amino acid , directed mutagenesis , secretory protein , biochemistry , gene , microbiology and biotechnology , recombinant dna
In an attempt to increase the secretion capacity of Pichia pastoris ( Pp ), PpPMR1 gene was disrupted with GS115 as parent strain, and the resultant mutant was designated as Pppmr1. Pppmr1 displayed a Ca 2+ ‐dependent growth defect, which was consistent with the PMR1 mutation in other yeasts. HSA–L5–IFNα2b, a human serum albumin (HSA) and inferferon‐α2b (IFNα2b) fusion protein with a flexible linker of 5 amino acid residues, was employed as a reporter to study the effects of PpPMR1 disruption on the secretion of heterologous protein. Because of its decreased viability after induction, Pppmr1 secreted more HSA–L5–IFNα2b only during the early phase (the first 15 hours) of induction. Although HSA–L5–IFNα2b secreted from GS115 and Pppmr1 had similar antiviral activity, the latter was heterogeneous (migrated as doublets on non‐reducing SDS–PAGE) and unstable (prone to aggregation at neutral to mild alkaline pH). Site‐directed mutagenesis revealed that the heterogeneity of HSA–L5–IFNα2b secreted from Pppmr1 was originated from the incomplete disulphide bridge pairing between Cys1 and Cys98 of IFNα2b. To be secreted homogeneously from Pppmr1 and to be stable in aqueous solution, the linker of the fusion protein should be extended to 10 amino acid residues. Copyright © 2008 John Wiley & Sons, Ltd.