Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis
Shakoori1,5, Muhammad Tariq Zahid,1,2 Farah Rauf Shakoori,3 Soumble Zulifqar,1 Ishtiaq Ahmed,4 Khalid Al-Ghanim,5 Shahid Mehboob,5 and Abdul Rauf . 2016
A new copper metallothionein (TfCuMT) gene has been identified from a locally isolated ciliate Tetrahymena farahensis. It contains 327
nucleotides encoding a peptide chain of 108 amino acids and belongs to class MTT2 and subfamily 7b. Amplification from both gDNA
and mRNA confirmed the intronless nature of this gene. Like most of the metallohtioneins, cysteine residues contribute nearly 30%
content with the specific CKC motifs. Structural repeats present in peptide sequence of TfCuMT indicate internal duplication of gene at
some stage of gene evolution. The predicted model of copper metallothionein protein showed that copper ions are mainly chelated by
thiol sulfur of cysteine residues and are embedded in the folds of polypeptide chain. For in vivo expression of TfCuMT in Escherichia
coli host cells the classical stop codons, which coded for glutamine in the ciliate were mutated to CAA and CAG through site directed
mutagenesis. The mutated gene showed higher expression in pET28a expression vector compared with pET21a. Optimum expression
was obtained after 6–8 h of 0.1mM IPTG induction. Stability of His tagged TfCuMT in 5% SDS was low, with half-life of about 104 min.
Presence of 1.0 mM copper increased the expression level by 1.65-fold. Presence of 100 mM Cysteine in culture medium caused 2.4-fold
increase in expression level. His tagged TfCuMT was purified through affinity chromatography using NTN-His binding resin in the
presence of 0.1M imidazole and NaCl. The modeled structure of the TfCuMT showed a cleft for Cu binding with correct orientation of
Cys residues in the motif CKC. J. Cell. Biochem. 117: 1843–1854, 2016. © 2016 Wiley Periodicals, Inc.
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