Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

Microbial Cell Factories

Research

Terrance A Stadheim¹  Byung-Kwon Choi¹  Dewan Andrews¹  Song Lin¹  Ming-Tang Chen¹  Ishaan Shandil¹ 

[1] GlycoFi, Biologics Discovery, Merck Research Laboratories, Merck & Co., Inc, 21 Lafayette Street, Suite 200, 03766, Lebanon, NH, USA;

关键词: Mating;    Diploid;    Pichia pastoris;    Strain stability;    Fermentation;    Recombinant protein expression;   

DOI  :  10.1186/1475-2859-11-91

 received in 2011-12-22, accepted in 2012-06-16,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundYeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes.ResultsBy using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences.ConclusionsTo the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.

【 授权许可】

CC BY   
© Chen et al.; licensee BioMed Central Ltd. 2012