应用Profinity eXact标签在真核表达系统中纯化EGFP蛋白

Application of Profinity eXact tag in purifying EGFP in eukaryotic expression system

目的研究Profinity eXact标签在真核蛋白表达系统中的纯化效果。方法通过PCR扩增Profinity eXact标签,构建中间载体pUC18ETagEGFP,构建Profinity eXact标签绿色荧光蛋白真核表达载体FUETagEGFP,采用DNA-磷酸钙方法转染293FT细胞,收集过量表达Profinity eXact标签绿色荧光蛋白的细胞,随后提取蛋白进行纯化,采用荧光检测EGFP蛋白结合、纯化效率,最后利用SDS-PAGE检测纯化蛋白的纯度。结果经PCR扩增、酶切和测序,Profinity eXact标签、中间载体pUC18ETagEGFP、Profinity eXact标签真核表达载体构建正确,经荧光显微镜镜下观察Profinity eXact标签真核表达载体转染293FT细胞效果良好,经荧光检测确定EGFP蛋白结合、纯化效率,经SDS-PAGE发现Profinity eXact标签在真核蛋白系统中纯化效果良好。结论 Profinity eXact标签在真核蛋白表达系统中能正常工作,并有较好的纯化效果。

This study is aimed to investigate the purifying efficacy of Profinity eXact tagged fusion protein in eukaryotic expression system.We utilized PCR to amplify Profinity eXact tag and construct vector pUC18ETagEGFP,with the aim of constructing eukaryotic expression vector FUETagEGFP.The constructed vector FUETagEGFP was then transfected into 293FT cells by DNA-calcium phosphate precipitate to overexpress Profinity eXact tagged EGFP proteins.Subsequently,the proteins were extracted and purified with Profinity eXact mini spin column.The purifying efficacy was analyzed by fluorescence detection and SDS-PAGE;the expressing of Profinity eXact tagging proteins were confirmed by fluorescent microscope.Moreover,the Profinity eXact tag could bind to immobilized subtilisin protease in Profinity eXact mini spin column and fluoride containing elution buffer triggers subtilisin to cleave the tag from the fusion proteins.Profinity eXact tag could be used in eukaryotic expression system and demonstrate well purifying efficacy.