Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica

Abstract

The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the NIT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein-S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione-Sepharose affinity chromatography column and digested with enterokinase, the NIT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd-MT and Zn-MT react with DTNB biphasically, and that Zn-MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu-MT reacts monophasically and releases metal slowly from MT. (c) 2006 Elsevier Inc. All rights reserved.