[Expression and purification of human metallothionein-1E gene in Escherichia coli].

Abstract

Objective: To express and purify human metallothionein-1E (MT-1E) fusion protein in vitro.

Methods: The cDNA encoding human MT-1E was amplified by RT-PCR and cloned into prokaryotic expressing vector pQE40. After transforming it into Escherichia Coli M15, we determined the solubility of target protein by western blotting and investigated the IPTG inducing condition. The target protein was purified from lysates with Ni-NTA agarose column.

Results: Western blotting analysis suggested that both the soluble and insoluble fusion protein existed in Escherichia Coli, but the insoluble was the main expression form. Induced by 1 mM IPTG, the expression of target protein increased with the prolongation of induction time. In our study, after being induced for 8h, the target protein accounted for about 32% of the total bacterial protein. Purified protein was obtained by affinity chromatography.

Conclusion: We have obtained purified human MT-1E fusion protein, which lays the foundation for the antibody preparation and further functional study.