哺乳动物极性蛋白mInscuteable-C末端肽段/GST融合蛋白的原核表达与纯化

Construction and Purification of a Prokaryotic Expression Vector of GST-mInsc257～532 Fusion Protein

目的:构建哺乳动物极性蛋白mInscuteable C末端257～532位氨基酸结构域与谷胱甘肽巯基转移酶(GST)的融合蛋白GST-mInsc 257～532的原核表达载体,在大肠杆菌中表达并纯化该融合蛋白。方法:将已经构建好的mInsc 257～532位氨基酸序列克隆至原核表达载体pGEX-4T中,构建重组的质粒pGEX-4T/mInsc 257～532;将重组质粒转化感受态细菌BL21,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达GST蛋白;经谷胱甘肽-琼脂糖球珠分离纯化;产物经SDS-PAGE电泳及Western Blot鉴定。结果:获得高表达及纯化的pGEX-4T/mInsc 257～532融合蛋白。结论:成功构建重组pGEX-4T/mInsc 257～532原核表达载体;诱导表达pGEX-4T/mInsc 257～532融合蛋白并纯化。

Objective. To construct a prokaryotic expression vector of GST-mInsc257~532 fusion protein and to express and purify the fusion protein in E. coli. Methods. A prior construc- ted mInscuteable fragment, the coding sequence of the C terminal of mInscuteable including the 257-532 amino acids, was cloned into pGEX-4T vector down stream of the GST tag to construct a recombinant plasmid pGEX-4T/mInsc257 - 532. Then the plasmid was transformed into E. col/BL21 and induced to express fusion protein GST/mInsc257-532 with IPTG. The recombi- nant GST-mlnsc257-532 fusion protein was purified by Glutathione Sepharose beads and then analyzed by SDS-PAGE and western blotting. Results. The highly expressed and purified pGEX- 4T/mInsc257-532 recombinant fusion protein was successfully obtained. Conclusion. The re- combinant plasmid pGEX-4T/mInsc257-532 was successfully constructed. GST/mInsc257-532 fusion protein was successfully expressed and purified.