title: Enhanced expression of recombinant elastase in Pichia pastoris through the substitution of Thr for Ser in Asn-Xaa-Ser sequons.
abstract: N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in Pichia pastoris, exerting great effects on expression efficiency; however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. At N212 or N280, substitution increased rPAE production effectively by 43 and 25 %, respectively. In comparison, at N36, N43, and N264, the change inhibited rPAE expression to varying extents; specifically, substitution at N36 resulted in a 31 % decrease, while substitution at N43 or N264 resulted in a decrease of less than 9 %. It is suggested that the effect of the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser on rPAE expression is roughly related to the role of the original Asn-Xaa-Ser sequon. As the conversion of Ser to Thr at N-glycosylation sites through site-directed mutagenesis is easily achieved, it is a feasible means of improving the expression of recombinant proteins in P. pastoris.
authors.affiliation: Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake; Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, School of Life Sciences, Huaiyin Normal University, Huaian, 223300, China, minghaihan2014@foxmail.com.
authors.forename: Minghai
authors.initials: M
authors.lastname: Han
authors.forename: Weixian
authors.initials: W
authors.lastname: Wang
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Cao
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tao
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Yu
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics*
chemical_list: D010196:Pancreatic Elastase