Prokaryotic expression and purification of human cytoskeleton regulation gene Nelin

Objective:To purify recombinant protein of human cytoskeleton regulation gene Nelin expressed in Escherichia coli.Methods:The B cell epitopes of human Nelin gene were predicted by DNAstar software. A cDNA fragment of Nelin gene with two epitopes was amplified by PCR, and expression plasmid vector pET28a(+) was ligated together with T_4DNA ligase after digested by corresponding restriction endonucleases respectively. The new constructed vector pET28a-Nelin was identified by endonuclease digesting and confirmed by DNA sequencing. The BL21(DE3) containing pET28a-Nelin plasmid was induced by IPTG, and the expressed protein were purified with Ni~ 2+ metal chelate affinity chromatography columns.Results:The recombinant expression vector pET28a-Nelin was successfully constructed. After induction, a new protein band of approximately 17 000 appeared on SDS-PAGE as soluble protein, which accounted for 18% of the total bacterial protein. Purified protein was obtained by Ni~ 2+ metal chelate affinity chromatography.Conclusion:The stability expression method of Nelin gene is set up, and the result lays a foundation for the study of Nelin gene function.