关键词: hTNFR1, 大肠杆菌, NusA标签, tig分子伴侣, 蛋白纯化

Abstract: The human tumor necrosis factor type 1 receptor (hTNFR1) gene was cloned into pET-22b expression vector, this study successfully constructed the recombinant plasmid pETH1 and transformed it into Escherichia coli BL21 (DE3) expression strain for shake flask fermentation, realizing heterologous expression of hTNFR1 in E. coli. However, all the proteins were in the forms of inclusion bodies. To improve the soluble expression of the target protein existed in the sedimentation in E. coli, we used two strategies of fusion tagging and molecular chaperone for protein folding. Results showed that, the fusion of NusA to the N-terminus of hTNFR1 increased the solubility; based on NusA-hTNFR1, seven molecular chaperones was overexpressed, among which the molecular chaperone tig was screened for it showed significant promotion on the soluble expression of hTNFR1 in E. coli; we purified protein by Ni-NTA affinity chromatography, then, the tig of NusA in the N-terminus of hTNFR1 was removed by enzymatic hydrolysis with TEV protease. After identification by Western blot, the hTNFR1 protein with high purity was obtained. The results laid a good foundation for further study on the physiological activity of hTNFR1 as well as the treatment of diseases.

Key words: hTNFR1, E. coli, NusA tag, tig molecular chaperone, protein purification