title: Engineering of a Pichia pastoris expression system for high-level secretion of HSA/GH fusion protein.
abstract: Human serum albumin (HSA) and human growth hormone (hGH) fusion protein [HSA/GH] is a promising long-acting form of GH to treat GH deficiency. This study attempted to engineer a P. pastoris strain for high-level production of HSA/GH to be used in basic research and clinical application. Strains contained two, three, and seven copies of HSA/GH gene were screened by selecting against Zeocin resistance. The results revealed that introducing two to three copies of HSA/GH gene was sufficient to give a significant increase in secretion level, compared with one copy of HSA/GH gene. No significant differences were observed between two to three copies and seven copies. Co-expression with either one copy of exogenous ERO1 or PDI in a strain carrying multicopies of HSA/GH gene led to varying degrees of increase in HSA/GH secretion. The effect of introducing multicopies of PDI was similar to that of one copy of PDI, but introducing excess copies of ERO1 reduced HSA/GH secretion. Simultaneous co-expression with PDI and ERO1 was less effect than either PDI co-expression or ERO1 co-expression. A strain showing higher secretion level was successfully applied to large-scale fermentation with the productivity of 3-4 g/l.
authors.affiliation: Institute of Pharmacology and Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.forename: Wenhui
authors.initials: W
authors.lastname: Liu
authors.forename: Guanghui
authors.initials: G
authors.lastname: Yang
authors.forename: Dengke
authors.initials: D
authors.lastname: Yu
authors.forename: Dianhai
authors.initials: D
authors.lastname: Lin
authors.forename: Hongying
authors.initials: H
authors.lastname: Sun
authors.forename: Shuqing
authors.initials: S
authors.lastname: Chen
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology; D005285:Fermentation; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012709:Serum Albumin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D001761:Bleomycin; D019382:Human Growth Hormone; C105427:Zeocin; D050862:Oxidoreductases Acting on Sulfur Group Donors; D019704:Protein Disulfide-Isomerases