大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化

doi:10.3724/SP.J.1005.2010.00505

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HEREDITAS ›› 2010, Vol. 32 ›› Issue (5): 505-511.doi: 10.3724/SP.J.1005.2010.00505

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Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli

WANG Zhong-Shan, XIANG Quan-Ju, WANG Hai-Yan, ZHANG Yi-Zheng

College of Life Sciences, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu 610064, China

Received:2009-12-11 Revised:2010-01-16 Online:2010-05-20 Published:2010-05-05
Contact: ZHANG Yi-Zheng E-mail:yizzhang@scu.edu.cn

Abstract

Abstract:

Cloning and expression of gsiB was carried out for studying protein structure and function of glutathione transport system. The coding sequence of Escherichia coli gsiB that encodes the periplasmic solute-binding protein of a glutathione transporter was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring GFP reporter gene through the method Sequence and Ligation–Independent Cloning (SLIC). The resulting recombinant plasmid pWaldo-GFP-GsiB was transformed into different E. coli strains and the expression conditions were optimized. It was found that E. coli BL21(DE3) was the best strain for gsiB gene expression among the four strains tested. Induction at lower incubation temperature of 18°C and 0.1 mmol/L of IPTG led to higher expression of gsiB in E. coli BL21(DE3). Western blotting analysis also showed the expression of gsiB and the molecular weight of expressed protein as expected.

Key words: Escherichia coli, glutathione transporter, periplasmic solute-binding component, gsiB, gene expression, Western blotting

WANG Zhong-Shan, XIANG Quan-Jie, WANG Hai-Yan, ZHANG Xi-Zheng. Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli[J]. HEREDITAS, 2010, 32(5): 505-511.

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Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli

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