title: Effects of co-overexpression of secretion helper factors on the secretion of a HSA fusion protein (IL2-HSA) in pichia pastoris.
abstract: Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2-HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2-HSA to ~2.3-, 1.9-, 2.2-, 2.5- and 1.9-fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: School of Food Science, Shihezi University, Shihezi, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Guan
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Fengxiang
authors.initials: F
authors.lastname: Chen
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Su
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Zuoying
authors.initials: Z
authors.lastname: Duan
authors.affiliation: Laboratory of Drug Design axnd Molecular Pharmacology, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Huazhong
authors.initials: H
authors.lastname: Li
authors.affiliation: Laboratory of Drug Design axnd Molecular Pharmacology, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
keywords: Pichia pastoris; SM protein; albumin fusion protein; chaperone; foldase; secretion helper factor