Title
Physiological Importance of Cadmium Metallothionein Protein: Cloning, Expression, Purification and Physiological Importance of M,Used
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TMCd1 is a stress inducible metallothionein (MT)gene that is expressed in Tetrahymena tropicalis lahorensis under cadmium metal stress. In the present study TMCd1 gene was expressed in E.coli and physiological importance of this Tetrahymena MT protein was evaluated. This stress inducible TMCd1 gene encodes 471 nucleotides, with TAA encoding glutamine (Gln) and TGA codes for a stop codon. To get the expression of full length protein the TMCd1 gene was mutated by site directed mutagenesis and TAA (at position 116) was converted to CAA (Gln). The MutTMCd1 gene was cloned in pET41a expression plasmid and MutTMCd1 protein was expressed as a fusion to glutathioneStransferase (GST) in E. coli BL21 CodonPlus (DE3) RIL cells. Upon induction with lactose the 48KDa MutTMCd1 fusion protein was expressed to a level of approximately 25% of total protein expressed in E. coli cells. Purification of MutTMCd1 protein by affinity chromatography resulted in a 48KDa band of fusion protein on 12% SDSPAGE. Cd2+ bioaccumulation experiments revealed that E. coli cells expressing the functional MutTMCd1 protein accumulated 19 fold higher cadmium as compared with control (without MutTMCd1 gene) cells.
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