Enhanced cadmium binding ability in response to novel modifications in a Paramecium cadmium metallothionein PMCd1

Metallothioneins (MTs) are low molecular weight cysteine rich proteins involved in detoxification of heavy metals. They are synthesized in response to metal exposure and can bind to various metals, thus reducing their toxicity and providing protection against oxidative stress. MTs are considered to be efficient bioremediators of heavy metal contaminated industrial wastewater. The present study was aimed at further enhancing the metal binding capacity of a cadmium metallothionein protein PMCd1, reported some time back from this laboratory in protozoan ciliate Paramecium, to equip them with more efficient system to deal with metal contaminated water bodies. Three additional cysteine residues were introduced at three different places of the protein by site directed mutagenesis, viz. S20C, R180C and Y185C. The wild type and each mutant of PMCd1 were expressed in E. coli BL21 cells. Metal uptake ability of each transformant was determined in the presence of 1 and 2mM Cd2+ in the medium. The three mutants showed enhanced metal uptake compared to the wild PMCd1 which underscored the role of additional cysteines in enhanced metal binding ability. Amongst the mutants, the genetically modified organism with S20C mutation exhibited 9.1 fold more metal uptake compared to the control ciliate. This mutant has great potential to clean the cadmium- contaminated water.