期刊
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
卷 40, 期 6, 页码 589-599出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s10295-013-1264-8
关键词
Heterologous protein expression; Proteolytic degradation; Pichia pastoris; Yapsin; Proteinase A
资金
- Science and Technology of Department of Zhejiang Province, China [2007C03001-2, 2010C13006]
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 similar to 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 pep4a-(3)yps1a-(3) double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4a-(3)yps1a-(3) double disruptant remained intact, in comparison to only 30 % with the wild-type strain.
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