文献类型：期刊文献

英文题名：Decrease of hirudin degradation by deleting the KEXI gene in recombinant Pichia pastoris

作者：Ni, Zhenhua[1];Zhou, Xiangshan[1];Sun, Xueqian[1];Wang, Ya[1];Zhang, Yuanxing[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2008

卷号：25

期号：1

起止页码：1

外文期刊名：YEAST

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000252869200001)】；

语种：英文

外文关键词：pop-in/pop-out; gene deletion; Pichia pastoris; hirudin degradation

摘要：Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop-in/pop-out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115Hir Delta kex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/1 in GS115Hir Delta kex1 while it was only 1.1 g/l in GS115Hir. Copyright (C) 2007 John Wiley & Sons, Ltd.