Functional characterization of a type 3 metallolthionein isoform (OsMTI-3a) from rice

Abstract

Metallothioneins (MTs) are low-molecular weight proteins with high Cys content and a high affinity for metals. Plant MTs are classified into four types based on the arrangement of Cys in their amino acid sequences. In the present study, the gene encoding OsMTI-3a, a type 3 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl β-d-1-thiogalactopyranoside, the OsMTI-3a was expressed as glutathione-S-transferase (GST)-tagged fusion protein. In comparison to control strain, the cells expressing GST-OsMTI-3a accumulated more Cd2+, Ni2+ and Zn2+ when they were grown in the medium containing CdCl2, NiCl2 or ZnSO4. The recombinant GST-OsMTI-3a was purified using affinity chromatography. The UV absorption spectra recorded after the reconstitution of the apo-protein with different metals confirmed that GST-OsMTI-3a was able to form complexes with Cd2+, Ni2+, and Zn2+. The reaction of the protein-metal complexes with 5-5-dithiobis (2-nitrobenzoic) revealed that the order of affinity of GST-OsMTI-3a toward different metals was Ni2+≥Cd2+>Zn2+>Cu2+. © 2014 Elsevier B.V..