Functional analysis of metallothionein MTT5 from Tetrahymena thermophila

Metallothioneins (MTs) constitute a superfamily of cysteine‐rich proteins that bind heavy‐metal ions by metal‐thiolate clusters. Five MT genes from Tetrahymena thermophila was subdivided into 7a ( MTT1 , MTT3 , and MTT5 ) and 7b ( MTT 2 and MTT4 ) subfamilies. In the study, MTT5 was knocked out in Tetrahymena . The mutant cells were sensitive to Cd 2+ and Pb 2+ but poorly sensitive to Cu + . In the MTT5 knockout cells, the expression levels of MTT1 and MTT3 were significantly up‐regulated under Cd 2+ and Pb 2+ stresses, whereas the expression levels of MTT2 and MTT4 were significantly up‐regulated under Cu + stress relative to those in the wild‐type cells. Furthermore, recombinant GST‐MTT5 was expressed in Escherichia coli/ pGEX‐ MTT5 and purified by affinity chromatography. Fluorescence quenching analysis showed that apoMTT5 can bind 8 Cd 2+ , 8 Pb 2+ , and 12 Cu + . The metal‐binding ability of the MTT5 complex followed the order of Pb 2+  > Cd 2+  > Cu + . Meanwhile, the half‐maximal inhibitory concentrations of the heavy‐metal ions for E. coli/ pGEX‐ MTT5 were as follows: Cu + (483.9 µM) > Pb 2+ (410.7 µM) > Cd 2+ (130.8 µM). The accumulation of Cd 2+ , Pb 2+ , and Cu + in the E. coli /pGEX‐ MTT5 was enhanced relative to that of E. coli /pGEX‐4T. Results suggested that different MTs functionally compensated in Tetrahymena , and MTT5 was a potential candidate for cadmium and lead bioremediation.