Genetic design of stable metal-binding biomolecules, oligomeric metallothioneins

Metallothionein (MT) is a suitable model for investigating molecular intera ctions relating to the handling of metals in cells. However, the production of functional MT proteins in microorganisms has been limited because of th e instability of MT-the thiol group of cysteine is easily oxidized and prot eolysis occurs. To increase the binding ability and to stabilize MT, we des igned genes for dimeric and tetrameric MT and the genes were successfully o verexpressed in Escherichia coli to generate functional oligomeric MTs. A h uman MT-H (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient hMT-II was ligated to create a dimer ic MT, from which a tetrameric MT was then constructed. The increased molec ular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resis tance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion b odies, which were dissolved with dithiothreitol, and the purified apo-MTs w ere reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT proteins exhibited both Cd and Zn binding activities tha t were, respectively, two and four times higher than those of the hMT-II mo nomer protein. These stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.