质底物结合蛋白gsiB基因的克隆及其表达条件的优化.pdfVIP

HEREDITAS (Beijing) 2010 5 , 32(5): 505―511 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2010.00505 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 王中山, 向泉桔, 王海燕, 张义正 , 610064 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。 大肠杆菌; 谷胱甘肽转运系统; 细胞周质底物结合蛋白; gsiB 基因; 基因表达; 蛋白质印迹 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 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 harbor- ing 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 strai