[Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli].
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 degrees 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.
Wang
Zhong-Shan
ZS
College of Life Sciences, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu 610064, China. wzs852@126.com
Xiang
Quan-Ju
QJ
Wang
Hai-Yan
HY
Zhang
Yi-Zheng
YZ
D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D030262:Soybean Proteins / Q000235:genetics*; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D030262:Soybean Proteins; C086860:glutathione transporter; D049452:Green Fluorescent Proteins; C553864:ALDH3-1 protein, soybean; D000444:Aldehyde Dehydrogenase