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Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium.
Ecotoxicology. 2014 Jan; 23(1):56-64.E

Abstract

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn(2+), Cu(2+) and Cd(2+) in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins.

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Publisher Full Text (DOI)

Authors+Show Affiliations

He Y
School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan, 030006, Shanxi, People's Republic of China.
Ma W
No affiliation info available
Li Y
No affiliation info available
Liu J
No affiliation info available
Jing W
No affiliation info available
Wang L
No affiliation info available

MeSH

Amino Acid SequenceAnimalsArthropod ProteinsBrachyuraCadmiumCopperEscherichia coliGene Expression RegulationGenetic VectorsMetallothioneinMetals, HeavyPolymerase Chain ReactionRecombinant ProteinsSequence AlignmentSmall Ubiquitin-Related Modifier ProteinsZinc

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24276409

Citation

He, Yongji, et al. "Expression of Metallothionein of Freshwater Crab (Sinopotamon Henanense) in Escherichia Coli Enhances Tolerance and Accumulation of Zinc, Copper and Cadmium." Ecotoxicology (London, England), vol. 23, no. 1, 2014, pp. 56-64.
He Y, Ma W, Li Y, et al. Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium. Ecotoxicology. 2014;23(1):56-64.
He, Y., Ma, W., Li, Y., Liu, J., Jing, W., & Wang, L. (2014). Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium. Ecotoxicology (London, England), 23(1), 56-64. https://doi.org/10.1007/s10646-013-1151-0
He Y, et al. Expression of Metallothionein of Freshwater Crab (Sinopotamon Henanense) in Escherichia Coli Enhances Tolerance and Accumulation of Zinc, Copper and Cadmium. Ecotoxicology. 2014;23(1):56-64. PubMed PMID: 24276409.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium. AU - He,Yongji, AU - Ma,Wenli, AU - Li,Yingjun, AU - Liu,Jinping, AU - Jing,Weixin, AU - Wang,Lan, Y1 - 2013/11/27/ PY - 2013/10/14/accepted PY - 2013/11/27/entrez PY - 2013/11/28/pubmed PY - 2014/8/29/medline SP - 56 EP - 64 JF - Ecotoxicology (London, England) JO - Ecotoxicology VL - 23 IS - 1 N2 - Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn(2+), Cu(2+) and Cd(2+) in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins. SN - 1573-3017 UR - https://www.unboundmedicine.com/medline/citation/24276409/Expression_of_metallothionein_of_freshwater_crab__Sinopotamon_henanense__in_Escherichia_coli_enhances_tolerance_and_accumulation_of_zinc_copper_and_cadmium_ DB - PRIME DP - Unbound Medicine ER -