铜绿假单胞菌SOS反应阻遏蛋白LexA的复性及活性研究

目的:对LexA蛋白复性方法进行优化，对复性后的LexA蛋白的生物学活性进行分析。方法:采用含有GSH/GSSG的缓冲液，一步稀释法对变性LexA蛋白进行复性，用镍离子亲合柱及阳离子柱层析法对复性后的LexA蛋白进行纯化，再以Sephadex G-25凝胶柱脱盐，采用非变性聚丙烯酰胺凝胶电泳和RP-HPLC法检测复性效果，Western blot法分析复性前后及经DTT处理后的LexA蛋白的免疫反应性，凝胶滞留电泳试验检测复性LexA蛋白与DNA的特异性结合能力。结果:复性后的LexA蛋白出现单体和多聚体的形式，多聚体是由单条肽链聚合而成。LexA单体和多聚体与兔抗LexA多克隆抗体均有较好的反应性。复性后的LexA蛋白能与SOS盒序列发生特异性结合。

The renaturation and activity study of LexA from Pseudomonas aeruginosa

Objective To optimize the renaturation procedure of denatured LexA, prepare the repressor LexA from Pseudomonas aeruginosa (PA), which have the satisfactory biologic activity. Methods The LexA was renatured by the GSH/GSSG dilution method, and the renatured protein were purified by Ni2+ chelate affinity chromatography and gel filtration chromatography, following desalination by Sephadex G-25 gel column. The renaturation result were detected by the native polyacrylamide gel electrophoresis and RP-HPLC.The immunological activity of all LexA proteins, including the denatured , renatured protein and the renatured protein that was treated with the DTT, were determined by Western blot. Results The renatured LexA appears both monomer and multimer, which is confirmed by the native polyacrylamide gel electrophoresis analysis and RP-HPLC. Gel retardation experiments shows that the renatured LexA have satisfactory biologic activity.