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.

Cellular and Molecular Basis of Heavy Metal-Induced Stress in Ciliates

“…MT was first discovered in the cortex of horse kidney as a cadmium-binding protein 46 . MTs are ubiquitous, hydro-philic, cytosolic, cysteine-rich, low molecular weight stable proteins 47 . They act as antioxidants by protecting against stress, DNA damage and apoptosis 47,48 .…”

“…MTs are ubiquitous, hydro-philic, cytosolic, cysteine-rich, low molecular weight stable proteins 47 . They act as antioxidants by protecting against stress, DNA damage and apoptosis 47,48 . MTs help in scavenging free hydroxyl and superoxide radicals, and this reaction rate is 340-fold higher than GSH 48 .…”

“…MTs help in scavenging free hydroxyl and superoxide radicals, and this reaction rate is 340-fold higher than GSH 48 . Important biological role of MTs is in detoxification of heavy metals by protecting against excess reactive heavy metal ions, free radical scavenging and in the maintenance of metal ion homeostasis 47,49 .…”

Functional analysis of metallothionein MTT5 from Tetrahymena thermophila

“…MTT2 and MTT4 are strongly induced by Cu and lowly induced by Cd . CuMTs have been reported from different Tetrahymena species . However, the relationship among these different MTs is unclear.…”

“…No intron exists in the coding sequence of Tetrahymena MT genes. Lysine (Lys) residues are always adjacent to cysteine in subfamily 7b, while in subfamily 7a this association is scarce . The expression levels of these MT genes also different from one another and respond differently to various stressors.…”