人PAK4基因原核表达载体的构建及其重组蛋白表达

Construction of Prokaryotic Plasmid of Human PAK4 Gene and Identification of Its Recombinant Protein

目的构建hPAK4的原核表达载体并诱导和鉴定其表达。方法pCAN2-PAK4质粒经XhoI和BamH I双酶切后,克隆至GST融合表达载体pGEX-5X-1,在E.coliBL21中诱导GST-hPAK4融合蛋白表达,并经W estern印迹鉴定结果。结果hPAK4编码序列已被克隆至GST融合表达载体pGEX-5X-1,双酶切鉴定片段大小为2.4kb,并在E.coliBL21中获得了诱导表达,蛋白的分子量为92KD。结论成功构建了hPAK4基因原核表达载体,诱导表达出GST-hPAK4融合蛋白。

Objective To construct prokaryotic expression plasmid of human p21-activated kinase 4 （hPAK4） gene and identify its recombinant protein expression induced by isopro-pyl-1-thio-b-Dgalactopyranoside （IPTG）. Methods The coding sequence of PAK4 gene was digested from the plasmid （ pCAN2-PAK4 ） with Xhol and BamHI enzymes, and cloned into pGEX-5X-1. The expression of GST-hPAK4 fusion protein was induced by IPTG and identified by Western blot. Results The coding sequence of hPAK4 gene was cloned into the pGEX-5X-1 plasmid which was transformed into E. coli BL21. The length of the fragment was 2.4kb, identified by double enzymes digestion. The expression of GST-hPAK4 fusion protein was induced by IPTG, and the molecular weigbt of protein was 92KD. Conclusion The recombinant prokaryotic plasmid was successfully constructed into pGEX-5X-1. The expression of GST-hPAK4 fusion protein was induced by IPTG and identified.