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 m etallothionein 1 (MT) to be characterized. A heterologous expression s ystem in E.coli has led to high yields of their pure zinc-complexed fo rms. The cadmium(II) binding properties of recombinant Zn-4-alpha MT a nd Zn-3-beta MT have been studied by electronic absorption and circula r dichroism. The former binds Cd(II) identically to alpha fragments ob tained 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(I I) leads to Cd-4-beta MT which, with the extra loading of Cd(II), unra vels to give rise isodichroically to Cd-9-beta MT. The effect of cadmi um-displaced Zn(II) ions and excess Cd(II) above the full metal occupa ncy of three has been studied using Chelex-100. The Cd-3-beta MT speci es is stable in the presence of this strong metal-chelating agent.