title: Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium.
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.
authors.affiliation: School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan, 030006, Shanxi, People's Republic of China.
authors.forename: Yongji
authors.initials: Y
authors.lastname: He
authors.forename: Wenli
authors.initials: W
authors.lastname: Ma
authors.forename: Yingjun
authors.initials: Y
authors.lastname: Li
authors.forename: Jinping
authors.initials: J
authors.lastname: Liu
authors.forename: Weixin
authors.initials: W
authors.lastname: Jing
authors.forename: Lan
authors.initials: L
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism; D003386:Brachyura / Q000235:genetics* / Q000378:metabolism; D002104:Cadmium / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D019216:Metals, Heavy / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D060829:Arthropod Proteins; D019216:Metals, Heavy; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D002104:Cadmium; D003300:Copper; D008668:Metallothionein; D015032:Zinc