[Expression and purification of recombinant metallothionein of Eriocheir sinensis in E. coli].

ABSTRACT

Metallothioneins (MT) are potential candidates for medicine development and application. For the purpose of expressing recombinant MT in E. coli, a crab MT cDNA cloned into pGEM-T was subcloned into pET-GST and then transformed into Escherichia Coli BL21. The fusion protein was proved to be expressed in both soluble and insoluble form by SDS-PAGE and western blot. Since metallothionein chelate metal ions, which may effects the physiological process of E. coli, caused the production of recombinant protein was lower than expected. Optimization of the ions content in the culture medium improved expression. The protein was purified by Zn2+ affinity chromatography, and rinsed off with high imidazole (1.5 M) which was the result of MT chelating instead of His-tag. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.