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Secretory expression, immunoaffinity purification and metal-binding ability of recombinant metallothionein (ShMT) from freshwater crab Sinopotamon henanense.
Ecotoxicol Environ Saf. 2019 Mar; 169:457-463.EE

Abstract

Metallothioneins (MTs) are a super-family of ubiquitous, low-molecular-weight, cysteine-rich and metal-binding proteins. They are thought to play a predominant role in mediating metal metabolism and antioxidation. However, the accurate functions of MTs remain unclear in the physiological processes due to native proteins deficiency and little information of their metal-binding character. Freshwater crab Sinopotamon henanense is a decapod crustacean widely distributed in northern China, in which only one MT isoform (ShMT) has been reported so far. In order to shed light on the accurate role of ShMT, a novel recombinant ShMT in native form was over-expressed by phoA secreted expression system in Escherichia coli (E. coli). Then the ShMT proteins were purified using a one-step gentle immunoaffinity chromatography with a polyol-responsive mAb (PR-mAb) to ShMT, which was generated by conventional hybridoma technology followed by ELISA-elution. The Zn-, Cu-, and Cd-ShMT complexes were prepared by recombinant synthesis in metal-enriched media and reconstitution with metal ions, respectively. Further analysis about metal-binding capacity showed recombinant ShMT has high ability to bind Zn, Cu and Cd metals, although the recombinantly expressed and reconstituted metal-ShMT complexes have different metal-to-protein stoichiometry. Moreover, the affinity of recombinant protein for metal ions has been analyzed using competitive reaction with 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB). The results demonstrated the affinity of recombinant ShMT for metals was as follows: Cu＞Cd＞Zn. In summary, the experimental procedure we have developed facilitates production of recombinant ShMT with native characteristics for further research and the study of metal-binding ability could help further clarify the accurate functions of ShMT.

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Publisher Full Text (DOI)

Authors+Show Affiliations

He Y
Institute of Agro-Products Processing Science and Technology, Shanxi Academy of Agricultural Sciences, 79 Longcheng Street, Taiyuan 030031, Shanxi Province, PR China; School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan 030006, Shanxi Province, PR China. Electronic address: heyongji_919@163.com.
Wang L
School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan 030006, Shanxi Province, PR China.
Ma W
School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan 030006, Shanxi Province, PR China.
Lu X
Biology Institute of Shanxi, 50 Shifan Street, Taiyuan 030006, Shanxi Province, PR China.
Li Y
Institute of Agro-Products Processing Science and Technology, Shanxi Academy of Agricultural Sciences, 79 Longcheng Street, Taiyuan 030031, Shanxi Province, PR China.
Liu J
School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan 030006, Shanxi Province, PR China.

MeSH

Amino Acid SequenceAnimalsBrachyuraChinaChromatography, AffinityEnzyme-Linked Immunosorbent AssayEscherichia coliGene Expression RegulationMetallothioneinMetals, HeavyProtein BindingProtein IsoformsRecombinant Proteins

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30472469

Citation

He, Yongji, et al. "Secretory Expression, Immunoaffinity Purification and Metal-binding Ability of Recombinant Metallothionein (ShMT) From Freshwater Crab Sinopotamon Henanense." Ecotoxicology and Environmental Safety, vol. 169, 2019, pp. 457-463.
He Y, Wang L, Ma W, et al. Secretory expression, immunoaffinity purification and metal-binding ability of recombinant metallothionein (ShMT) from freshwater crab Sinopotamon henanense. Ecotoxicol Environ Saf. 2019;169:457-463.
He, Y., Wang, L., Ma, W., Lu, X., Li, Y., & Liu, J. (2019). Secretory expression, immunoaffinity purification and metal-binding ability of recombinant metallothionein (ShMT) from freshwater crab Sinopotamon henanense. Ecotoxicology and Environmental Safety, 169, 457-463. https://doi.org/10.1016/j.ecoenv.2018.11.065
He Y, et al. Secretory Expression, Immunoaffinity Purification and Metal-binding Ability of Recombinant Metallothionein (ShMT) From Freshwater Crab Sinopotamon Henanense. Ecotoxicol Environ Saf. 2019;169:457-463. PubMed PMID: 30472469.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Secretory expression, immunoaffinity purification and metal-binding ability of recombinant metallothionein (ShMT) from freshwater crab Sinopotamon henanense. AU - He,Yongji, AU - Wang,Lan, AU - Ma,Wenli, AU - Lu,Xiaoxia, AU - Li,Yunlong, AU - Liu,Jinping, PY - 2018/08/28/received PY - 2018/11/14/revised PY - 2018/11/16/accepted PY - 2018/11/26/pubmed PY - 2019/2/1/medline PY - 2018/11/26/entrez KW - Immunoaffinity purification KW - Metal-binding ability KW - Metallothionein KW - Polyol-responsive KW - Secretory expression KW - Sinopotamon henanense SP - 457 EP - 463 JF - Ecotoxicology and environmental safety JO - Ecotoxicol Environ Saf VL - 169 N2 - Metallothioneins (MTs) are a super-family of ubiquitous, low-molecular-weight, cysteine-rich and metal-binding proteins. They are thought to play a predominant role in mediating metal metabolism and antioxidation. However, the accurate functions of MTs remain unclear in the physiological processes due to native proteins deficiency and little information of their metal-binding character. Freshwater crab Sinopotamon henanense is a decapod crustacean widely distributed in northern China, in which only one MT isoform (ShMT) has been reported so far. In order to shed light on the accurate role of ShMT, a novel recombinant ShMT in native form was over-expressed by phoA secreted expression system in Escherichia coli (E. coli). Then the ShMT proteins were purified using a one-step gentle immunoaffinity chromatography with a polyol-responsive mAb (PR-mAb) to ShMT, which was generated by conventional hybridoma technology followed by ELISA-elution. The Zn-, Cu-, and Cd-ShMT complexes were prepared by recombinant synthesis in metal-enriched media and reconstitution with metal ions, respectively. Further analysis about metal-binding capacity showed recombinant ShMT has high ability to bind Zn, Cu and Cd metals, although the recombinantly expressed and reconstituted metal-ShMT complexes have different metal-to-protein stoichiometry. Moreover, the affinity of recombinant protein for metal ions has been analyzed using competitive reaction with 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB). The results demonstrated the affinity of recombinant ShMT for metals was as follows: Cu＞Cd＞Zn. In summary, the experimental procedure we have developed facilitates production of recombinant ShMT with native characteristics for further research and the study of metal-binding ability could help further clarify the accurate functions of ShMT. SN - 1090-2414 UR - https://www.unboundmedicine.com/medline/citation/30472469/Secretory_expression_immunoaffinity_purification_and_metal_binding_ability_of_recombinant_metallothionein__ShMT__from_freshwater_crab_Sinopotamon_henanense_ DB - PRIME DP - Unbound Medicine ER -