Evaluation of physiological importance of metallothionein protein expressed by <i>Tetrahymena cadmium metallothionein 1 (TMCd1)</i> gene in <i>Escherichia coli</i>

Suleman, Aliya; Shakoori, A. R.

doi:10.1002/jcb.24030

Cited by 8 publications

(9 citation statements)

References 15 publications

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“…MTs are more stable and less prone to proteolytic cleavage in metal bound state as compared to unbound state. The stable expression of MT protein was achieved when the cadmium was added to the cell cultures . Bioaccumulation experiments revealed that the E. coli cells expressing the TMCd1 protein accumulates cadmium at 7.9 μM/mg of cell wet weight .…”

Section: Discussionmentioning

confidence: 99%

“…The stable expression of MT protein was achieved when the cadmium was added to the cell cultures . Bioaccumulation experiments revealed that the E. coli cells expressing the TMCd1 protein accumulates cadmium at 7.9 μM/mg of cell wet weight . The induced E. coli /pET‐TMCd1 cells accumulated Cd 2+ at a 19‐fold higher amount than that of the control cells lacking the TMCd1 gene.…”

Section: Discussionmentioning

confidence: 99%

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Functional analysis of metallothionein MTT5 from Tetrahymena thermophila

Zhou

Wang

2018

J of Cellular Biochemistry

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Metallothioneins (MTs) constitute a superfamily of cysteine-rich proteins that bind heavy-metal ions by metal-thiolate clusters. Five MT genes from Tetrahymena thermophila was subdivided into 7a (MTT1, MTT3, and MTT5) and 7b (MTT2 and MTT4) subfamilies. In the study, MTT5 was knocked out in Tetrahymena. The mutant cells were sensitive to Cd and Pb but poorly sensitive to Cu . In the MTT5 knockout cells, the expression levels of MTT1 and MTT3 were significantly up-regulated under Cd and Pb stresses, whereas the expression levels of MTT2 and MTT4 were significantly up-regulated under Cu stress relative to those in the wild-type cells. Furthermore, recombinant GST-MTT5 was expressed in Escherichia coli/pGEX-MTT5 and purified by affinity chromatography. Fluorescence quenching analysis showed that apoMTT5 can bind 8 Cd , 8 Pb , and 12 Cu . The metal-binding ability of the MTT5 complex followed the order of Pb > Cd > Cu . Meanwhile, the half-maximal inhibitory concentrations of the heavy-metal ions for E. coli/pGEX-MTT5 were as follows: Cu (483.9 µM) > Pb (410.7 µM) > Cd (130.8 µM). The accumulation of Cd , Pb , and Cu in the E. coli/pGEX-MTT5 was enhanced relative to that of E. coli/pGEX-4T. Results suggested that different MTs functionally compensated in Tetrahymena, and MTT5 was a potential candidate for cadmium and lead bioremediation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Functional analysis of metallothionein MTT5 from Tetrahymena thermophila

Zhou

Wang

2018

J of Cellular Biochemistry

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“…Mutagenesis resulted in expression of a full length protein which is useful to characterize the protein and analyze its metal binding capacity. Suleman and Shakoori [] have also reported the functional analysis of a full length expressed mutated cadmium MT.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis resulted in expression of a full length protein which is useful to characterize the protein and analyze its metal binding capacity. Suleman and Shakoori [2012] have also reported the functional analysis of a full length expressed mutated cadmium MT. EXPRESSION AND PURIFICATION OF TfCuMT E. coli BL21C þ host cells were transformed with mutated gene cloned in pET21a expression vector for expression purpose.…”

Section: Mutagenesis Of Tfcumtmentioning

confidence: 99%

Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis

Zahid

Shakoori

Zulifqar

et al. 2016

J of Cellular Biochemistry

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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.

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“…Some studies reported that the prokaryotic expression could be applied to researching the eukaryotic gene functions (Evans et al . ; Suleman and Shakoori ). To further elucidate the relationship between AtLCD and AtDCD genes and tolerance to heavy metal, we cloned these two genes.…”

Section: Introductionmentioning

confidence: 98%

Cadmium toxicity is alleviated by AtLCD and AtDCD inEscherichia coli

et al. 2012

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Aims: Arabidopsis thaliana L-and D-cysteine desulfhydrases (AtLCD and AtDCD) are two important H 2 S-generating enzymes. This study determined the effects of H 2 S derived from AtLCD and AtDCD on cadmium (Cd) toxicity in Escherichia coli. Methods and Results: AtLCD and AtDCD were cloned into pET28a vectors and transformed into wild-type E. coli strain BL21(DE3), named BL21(LCD) and BL21(DCD). In the induced BL21(LCD) and BL21(DCD) compared with wild type, significantly higher H 2 S generation rates were observed. Additionally, higher survival rates, reduced contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ), decreased activities of superoxide dismutase and catalase under 220 lmol l À1 Cd stress were noted. We obtained similar results in the wild type treated with NaHS, a H 2 S donor. The above changes were substantially counteracted by the mixture of ammonia and pyruvic acid potassium (NH 3 + C 3 H 3 KO 3 ), a synthetic inhibitor of H 2 S. Conclusions: AtLCD and AtDCD catalyse the H 2 S production, generating an ameliorating effect against Cd-induced oxidative stress and resulting in E. coli resistance to Cd toxicity. Significance and Impact of the Study: H 2 S as a gasotransmitter is certified to have an ameliorating effect against Cd toxicity, thus providing information for further research regarding the role of H 2 S in regulating resistance to the heavy metal stress in organisms.

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Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia coli

Cited by 8 publications

References 15 publications

Functional analysis of metallothionein MTT5 from Tetrahymena thermophila

Functional analysis of metallothionein MTT5 from Tetrahymena thermophila

Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis

Cadmium toxicity is alleviated by AtLCD and AtDCD inEscherichia coli

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