Cloning and expression of a seed-specific metallothionein-like protein from sesame.

ABSTRACT

A cDNA clone, SiMT encoding an Ec type of metallothionein (MT)-like protein, was isolated from maturing seeds of sesame (Sesamum indicum L.), and its deduced protein sequence shared 47-65% similarity to other known Ec type of MT-like proteins with three highly conserved cysteine-rich segments. The transcript of SiMT was exclusively accumulated in maturing seeds from two weeks after flowering to the end of seed maturation. The results of a southern blot analysis suggested that one SiMT and one SiMT-like gene were present in the sesame genome. Recombinant SiMT fused with glutathione-S-transferase (GST) was over-expressed in Escherichia coli, and purified to homogeneity by affinity chromatography. Recombinant SiMT released from GST was harvested after factor Xa cleavage. Migration of the recombinant SiMT during SDS-PAGE was accelerated when its binding metal ions were depleted by EDTA. The metal-binding capability of recombinant SiMT was measured by inductively-coupled plasma atomic emission spectrometry. Our results show that the recombinant SiMT could trap zinc or copper ions, but not manganese ions, with a stoichiometric ratio (metal ion/SiMT) of approximately 2.