Recombinant synthesis of mouse Zn3-beta and Zn4-alpha metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectroscopic analyses, has enabled the metal binding properties of the alpha and beta subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn-4-alpha MT and Zn-3-beta MT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to alpha fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the native protein. Titration of Zn-3-beta MT with CdCl2 results in the formation of Cd-3-beta MT. The addition of excess Cd(II) leads to Cd-4-beta MT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd-9-beta MT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd-3-beta MT species is stable in the presence of this strong metal-chelating agent.