title: Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115.
abstract: Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.
authors.affiliation: Institute of Pharmacology and Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, Zhejiang, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.forename: Qi
authors.initials: Q
authors.lastname: Shen
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Wen
authors.initials: W
authors.lastname: Qu
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Hongying
authors.initials: H
authors.lastname: Sun
authors.forename: Shuqing
authors.initials: S
authors.lastname: Chen
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D010281:Parathyroid Hormone / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012709:Serum Albumin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; C470125:PTH protein, human; D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II