Cloning and expression of <i>Saccharomyces cerevisiae</i> copper‐metallothionein gene in <i>Escherichia coli</i> and characterization of the recombinant protein

Abstract

The gene sequences for intact and truncated forms of copper-binding metallothionein from Saccharomyces cerevisiae were cloned and overexpressed in Escherichia coli BL21(DE3)pLysE cells. In contrast to several other genes, the intact and truncated metallothionein genes are amplified in the polymerase chain reaction when Mg2+ is replaced by Co2+. The recombinant truncated protein binds copper in vivo and in vitro. A ratio of 8 Cu/12 cysteines was determined from atomic absorption, X-ray fluorescence and amino acid analysis. Extended X-ray absorption spectroscopy indicates that all Cu is in Cu(I) form and coordinated to three S atoms.