A synthetic DNA coding for human metallothionein II (hMT-II) was designed for efficient expression in Escherichia coli and cloned into a vector, pUEX2. Upon induction by heating, the gene for hMT-II was expressed as a fusion protein between β-galactosidase and hMT-II. The fusion protein accounted for approximately 24% of the total protein between β-galactosidase and hMT-II. The fusion protein accounted for approximatedly protein with a molecular mass of approximately 122 kDa was purified in homogeneity by washing the inclusion bodies with a solution of Triton X-100. The insoluble protein was easily solubilized in the presence of 2-mercaptoethanol and sodium dodecyl sulfate without any need for denaturants, such as urea or guanidine hydrochloride. The fused hMT-II protein bound to radioactive cadmium. A competition experiment with radioactive cadmium and various heavy metals showed that the hMT-II fusion protein had a higher affinity for Cu2+, Co2+, and Ag+ ions than Cd2+ and Zn2+ ions. Thus, we succeeded in over expression of hMT-II as an active form in E. coli.
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology