文献类型：期刊文献

英文题名：Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris

作者：Zhu, T.[1];Guo, M.[1,2];Tang, Z.[1];Zhang, M.[1];Zhuang, Y.[1];Chu, J.[1];Zhang, S.[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Jiangxi Agr Univ, Dept Biotechnol, Nanchang, Peoples R China

年份：2009

卷号：107

期号：3

起止页码：954

外文期刊名：JOURNAL OF APPLIED MICROBIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000268854000026)】；

基金：This research was supported by National Natural Science Foundation of China (no. 30560006), National Basic Research Program of China (973 Program, no. 2007CB714303), and High-tech Research Plan of China (863) (2007AA100601).

语种：英文

外文关键词：gene dosage; multi-copy Pichia pastoris; physiological performance; porcine insulin precursor

摘要：Aims: This study attempted to fully explore the expression potentials of Pichia pastoris for producing porcine insulin precursor (PIP) through PIP copy number optimization. Methods and Results: Multi-copy strains were screened employing a highly efficient improved in vivo method and their copy numbers were quantified by real-time qPCR. A range of Mut(+)P. pastoris strains harbouring 0, 1, 3, 6, 12, 18, 29, 52 copies of PIP were obtained. After 96 h methanol induction, a bell-shaped correlation curve was observed between gene dosage and protein yield, and the maximum PIP expression level of 181 mg l(-1) was achieved by a 12-copy strain. Specific growth rate and methanol utilization capacity were found to decrease remarkably for high copy strains (> 12 copies). Transcriptional analysis of KAR2 suggested higher copy strains were suffering more from ER stress. Conclusions: A copy number around 12 is optimal for secretory expression of PIP in P. pastoris. Excess PIP gene dosage (> 12 copies) significantly impaired the growth of P. pastoris hosts. Significance and Impact of the Study: The methods developed and the discoveries made by this systematical investigation will be helpful to the application and understanding of Pichia pastoris expression system for heterologous overexpression.