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Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis
Author(s) -
Zahid Muhammad Tariq,
Shakoori Farah Rauf,
Zulifqar Soumble,
Ahmed Ishtiaq,
AlGhanim Khalid,
Mehboob Shahid,
Shakoori Abdul Rauf
Publication year - 2016
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.25484
Subject(s) - metallothionein , tetrahymena , microbiology and biotechnology , gene , cysteine , biochemistry , biology , gene expression , amino acid , peptide , expression vector , escherichia coli , peptide sequence , chemistry , affinity chromatography , recombinant dna , enzyme
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 Tf CuMT 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 0.1 mM IPTG induction. Stability of His tagged Tf CuMT in 5% SDS was low, with half‐life of about 104 min. Presence of 1.0 μM copper increased the expression level by 1.65‐fold. Presence of 100 μM Cysteine in culture medium caused 2.4‐fold increase in expression level. His tagged Tf CuMT was purified through affinity chromatography using NTN‐His binding resin in the presence of 0.1 M 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.