Design of Artificial Polypeptides That Bind a Large Number of Heavy Metal Ions by Use of a Cyanobacterial Metallothionein

Abstract

SmtA, a metallothionein of the cyanobacterium Synechococcus sp. PCC 7942, binds heavy metal ions such as Zn²⁺ and is involved in regulation of the homeostasis of Zn²⁺. NMR analysis has indicated that SmtA has two metal-binding regions, namely, the N-terminal region (Met1-Cys47) and C-terminal region (Gly43-Gly56), which binds two and one molecules of Zn²⁺, respectively. We attempted to improve the binding capacity for Zn²⁺ by jointing several N-terminal or C-terminal regions tandemly by genetic engineering. The recombinant proteins fused with a GST were overexpressed in E.coli and purified. ICP-spectrometry analysis revealed that the binding number of Zn²⁺ increased by two in proportion to one N-terminal region and by one to one C-terminal region. Strikingly, a recombinant SmtA with additional 10 N-terminal regions bound 23 molecules of Zn²⁺. Tandem joint of the C-terminal region improved the binding efficiency of Zn²⁺ per the number of amino acid residue.