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.[1,2,3]; Tang, Z.[1]; Zhang, M.[1]; Zhuang, Y.[1]; Chu, J.[1]; Zhang, S.[1]

机构：[1] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China; [2] Department of Biotechnology, Jiangxi Agricultural University, Nanchang, China; [3] East China University of Science and Technology, 130 Meilong Rd., Shanghai 200237, 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：EI(收录号：20231713997051)

语种：英文

外文关键词：Insulin - Methanol - Polymerase chain reaction - Strain rate - Yeast

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. ? 2009 The Society for Applied Microbiology.