College of Life Sciences, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu, China.
Genet Mol Biol. 2011 Oct;34(4):661-8. doi: 10.1590/S1415-47572011005000043. Epub 2011 Oct 1.
The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.
谷胱甘肽（硫）输入系统 ATP 结合蛋白（gsiA）的克隆、表达和纯化。通过 PCR 扩增编码大肠杆菌 gsiA 的编码序列，gsiA 编码谷胱甘肽输入体的 ATP 结合蛋白，然后将其插入带有绿色荧光蛋白（GFP）报告基因的原核表达载体 pWaldo-GFPe 中。所得重组质粒 pWaldo-GFP-GsiA 转化为各种大肠杆菌菌株，并优化表达条件。评估了五种大肠杆菌表达菌株对重组 gsiA 蛋白产生的影响。发现大肠杆菌 BL21（DE3）是 GsiA-GFP 融合蛋白表达最具生产力的菌株，其中大部分是不溶性部分。然而，胶内和 Western blot 分析的结果表明，在 Rosetta（DE3）中表达重组 GsiA 以可溶形式提供了有效的来源。通过使用 GFP 作为报告基因，方便地获得了最合适的宿主菌株，从而优化了蛋白质的过表达和纯化条件，不仅使进一步的功能和结构研究更加省力，而且节省了时间。
