绿色荧光蛋白标记的表达载体pHis-EGFP的构建

Construction of an Expression Vector pHis-EGFP with the Enhanced Green Fluorescent Protein (EGFP) Gene

为对重组蛋白的表达进行直观检测并简化蛋白纯化的步骤,构建了能在大肠杆菌中表达融合蛋白的通用表达载体pHis-EGFP。该载体含有源自表达载体pET-32a的T7启动子、终止子和源自质粒pUC18的ColE1复制子与绿色荧光蛋白报告基因。应用该载体成功地表达并纯化了酵母GGDP(geranylgeranyldiphosphate,GGDP)合酶融合蛋白,结果表明所构建的载体是一个实用的表达载体,并建立了离子交换层析和亲和层析两步纯化融合蛋白的方法。

The plasmid pHis-EGFP is a expression vector based on the plasmid pUC 18. It was constructed by assembling a T7 operator region, the 6 × His tag coding sequence, the multiple cloning site, and T7 terminator from plasmid pET-32a（ ＋ ） with the ColE 1 origin of the replicon and the fragment of the enhanced green fluorescent protein gene （EGFP） from pEGFP-N1. The constructed vector could be used to express the His-tagged, EGFP-labeled fusion proteins in Escherichia coli. Thus, the recombinant protein will be easy to detect and purified. With the constructed vector the geranylgeranyl diphosphate synthase of yeast has been successfully expressed in E. coli and purified. The result indicates that the constructed vector is effective and practicable. A simple, 2-step method of protein purification was developed by the combination of ion exchange and affinity chromatography.