title: Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis.
abstract: 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 0.1 mM IPTG induction. Stability of His tagged TfCuMT 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 TfCuMT 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.
authors.affiliation: School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan.|Departmenmt of Zoology, GC University, Katchery Road, Lahore, Pakistan.
authors.forename: Muhammad Tariq
authors.initials: MT
authors.lastname: Zahid
authors.affiliation: Departmenmt of Zoology, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan.
authors.forename: Farah Rauf
authors.initials: FR
authors.lastname: Shakoori
authors.affiliation: School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan.
authors.forename: Soumble
authors.initials: S
authors.lastname: Zulifqar
authors.affiliation: Institute of Industrial Biotechnology, GC University, Katchery Road, Lahore, Pakistan.
authors.forename: Ishtiaq
authors.initials: I
authors.lastname: Ahmed
authors.affiliation: Department of Zoology, College of Science, King Saud University, PO Box 24555, Riyadh 11451, Kingdom of Saudi Arabia.
authors.forename: Khalid
authors.initials: K
authors.lastname: Al-Ghanim
authors.affiliation: Department of Zoology, College of Science, King Saud University, PO Box 24555, Riyadh 11451, Kingdom of Saudi Arabia.
authors.forename: Shahid
authors.initials: S
authors.lastname: Mehboob
authors.affiliation: School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan.|Department of Zoology, College of Science, King Saud University, PO Box 24555, Riyadh 11451, Kingdom of Saudi Arabia.
authors.forename: Abdul Rauf
authors.initials: AR
authors.lastname: Shakoori
mesh_terms: D017125:Genes, Protozoan; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D055550:Protein Stability; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013768:Tetrahymena / Q000235:genetics / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D008668:Metallothionein
keywords: AFFINITY CHROMATOGRAPHY; COPPER; COPPER METALLOTHIONEIN; CYSTEINE; IPTG INDUCTION; MTT2 FAMILY OF METALLOTHIONEIN; SITE DIRECTED MUTAGENESIS; TETRAHYMENA FARAHENSIS; TfCuMT GENE