基于Profinity eXact系统的可溶性egfp表达和纯化

Prokaryotic Expression and Purification of Soluble egfp Based on the Profinity eXact System

为研究Profinity eXact系统在蛋白表达及纯化过程中的效果,利用PCR扩增绿色荧光蛋白基因egfp,定向克隆至表达载体pPAL7上,转化BL21(DE3),荧光显微镜下观察诱导后的重组菌;取超声破碎的上清挂柱纯化,紫外下检测纯化后egfp发出荧光的特性,Western blot分析其免疫反应性。结果表明:诱导后的重组菌pPAL7-egfp/BL21(DE3)在紫外光下能够发出绿色荧光;一步纯化后的egfp蛋白同样也能在紫外激发下发出绿色荧光,同时egfp蛋白能和特异性抗体结合,具有良好的免疫反应性。实验结果说明Profinity eXact系统对于可溶性蛋白的表达和纯化,方法操作简单、快捷,具有很好的应用价值。

To study the use of Profinity eXact system in the expression and purification for the egfp protein,Egfp gene was applied by PCR and inserted into the pPAL7 vector. Recombinant bacteria pPAL7-egfp/BL21（DE3） was constructed for expression. Then fluorescence microscope and SDS-PAGE were used to investigate the egfp protein expression and purification with Profinity eXact system. Green fluorescence of the induced recombinant bacteria was observed under fluorescence microscope, and fluorescence of one step purified egfp could be detected too. The purified egfp could be recognized by anti-GFP antibody in Western blot, which demonstrated the good immunoreactions of protein egfp. In conclusion, the results showed Profinity eXact system was very simple and convenient for purification of soluble protein, so it had good application value in such area.