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

为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能, 对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列, 利用PCR方法扩增到该基因的编码区序列, 利用SLIC (Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中, 成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达, 通过改变培养温度和IPTG浓度等条件, 得到了能够大量表达目标蛋白的重组子。结果表明: 大肠杆菌BL21(DE3)是 gsiB基因表达的最佳宿主菌; 18℃低温诱导培养有利于gsiB基因的大量表达; 0.1 mmol/L IPTG足够诱导gsiB基因表达, 增加IPTG浓度(0.1 mmol/L~1.0 mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达, 其分子量大小与预期相符。

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°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.