High-level Expression of a Type 2 Metallothionein Protein（HcMT）from Halostachys caspica in Escherichia coli and Its Metal-binding Ability

doi:10.13560/j.cnki.biotech.bull.1985.2016.04.016

Abstract

Abstract: This study aims to evaluate the prokaryotic expression and metal-binding ability of metallothionein protein in Halostachys caspica（HcMT）. HcMT cloned from H. caspica was transferred to prokaryotic expression vector pGEX-6p-2 and expressed in Escherichia coli BL21. Then the glutathione S-transferase（GST）fusion protein（GST-HcMT）was isolated and purified. Using atomic absorption spectrometry，the metal-binding ability of the GST-HcMT was determined by measuring the amount of metal ions it bound with. The results showed that the molecular weight of expressed fusion protein GST-HcMT was about 34 kD，which was as expected and confirmed by Western blot. Furthermore，GST-HcMT bound 25，10，4，2 folds of Cu2+，Zn2+，Pb2+，and Cd2+ separately as GST alone，and the binding ability of GST-HcMT was as follows：Cu2+ > Cd2+ > Zn2+ > Pb2+.

Key words: metallothionein protein from Halostachys caspica（HcMT）, atomic absorption spectrometry（AAS）, metal-binding ability

MENG Hong-en, DU Rong-feng, XU Xin,LIU Zhong-yuan. High-level Expression of a Type 2 Metallothionein Protein（HcMT）from Halostachys caspica in Escherichia coli and Its Metal-binding Ability[J]. Biotechnology Bulletin, 2016, 32(4): 121-127.

References

［1］Jin S, Cheng Y, Guan Q, et al. A metallothionein-like protein of rice（rgMT）functions in E. coli and its gene expression is induced by abiotic stresses［J］. Biotechnol Lett, 2006, 28（21）：1749-1753.
［2］Hassinen VH, Tervahauta AI, Schat H, et al. Plant metallothioneins- metal chelators with ROS scavenging activity?［J］. Plant Biology, 2011, 13（2）：225-232.
［3］Leszczyszyn OI, Imam HT, Blindauer CA. Diversity and distribution of plant metallothioneins：a review of structure, properties and functions［J］. Metallomics, 2013, 5（9）：1146-1169.
［4］Capdevila M. Metallothionein protein evolution：a miniassay［J］. J Biol Inorg Chem, 2011, 16（7）：977-989.
［5］Usha B, Venkataraman G, Parida A. Heavy metal and abiotic stress inducible metallothionein isoforms from Prosopis juliflora（SW）D.
［6］ Freisinger E. Structural features specific to plant metallothioneins［J］. J Biol Inorg Chem, 2011, 16（7）：1035-1045.
［7］Grennan AK . Metallothioneins, a diverse protein family［J］. Plant Physiol, 2011：155.
［8］Mir G. A plant type 2 metallothionein（MT）from cork tissue responds to oxidative stress［J］. J Eep Bot, 2005, 55（408）：2483-2493.
［9］ Shunsaku N, Liu S, Tetsuo T. Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Sacch-aromyces cerevisiae［J］. Biotechnol Lett, 2007, 29（8）：1301-1305.
［10］ Yang Z, Wu Y, Li Y, et al. OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice［J］. Plant Mol Biol, 2009, 70（1-2）：219-229.
［11］Yang J, Wang Y, Liu G, et al. Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd2+, Zn2+, Cu2+, and NaCl in transgenic yeast［J］. Mol Biol Rep, 2011, 38（3）：1567-1574.
［12］Foley RC, Liang ZM, Singh KB. Analysis of type 1 metallothionein cDNAs in Vicia faba［J］. Plant Mol Biol, 1997, 33（4）：583-591.
［13］Huang GY, Wang YS. Expression and characterization analysis of type 2 metallothionein from grey mangrove species（Avicennia marina）in response to metal stress［J］. Aquat Toxicol, 2010, 99（1）：86-92.
［14］Huang GY, Wang YS, Ying GG. Cadmium-inducible BgMT2, a type 2 metallothionein gene from mangrove species（Bruguiera gymnorrhiza）, its encoding protein shows metal-binding ability［J］. J Exp Mar Biol Ecol, 2011, 405（1-2）：128-132.
［15］An ZG, Li CJ, Zu YG, et al. Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings［J］. J Exp Bot, 2006, 57（14）：3575-3582.
［16］Hassinen VH, Tuomainen M, Per?niemi S, et al. Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyper accumulator, Thlaspi caerulescens［J］. J Exp Bot, 2009, 60（1）：187-196.
［17］Veerle MJG, Barbara I, Henk WJH, et al. Expression of the Arabidopsis metallothionein 2b enhances arsenite sensitivity and root to shoot translocation in tobacco［J］. Environ Exp Bot, 2009, 66（1）：69-73.
［18］Young OA, Sun HK, Jeongyeo L, et al. Three Brassica rapa metallothionein genes are differentially regulated under various stress conditions［J］. Mol Biol Rep, 2012, 39（3）：2059-2067.
［19］Sereno ML, Almeida RS, Nishimura DS, et al. Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes［J］. J Plant Physiol, 2007, 164（11）：1499-1515.
［20］Yuan J. Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice［J］. Plant Physiol, 2008, 146（4）：1637-1650.
［21］Xue T, Li X, Zhu W, et al. Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast［J］. J Exp Bot, 2009, 60（1）：339-349.
［22］Murphy A. Purification and Immunological Identification of Metallothioneins 1 and 2 from Arabidopsis thaliana［J］. Plant Physiol, 1997, 113（4）：1293-1301.
［23］Abdullah SNA, Cheah S, Murphy DJ. Isolation and characterisation of two divergent type 3 metallothioneins from oil palm, Elaeis guineensis［J］. Plant Physiol Biochem, 2002, 40（3）：255-263.
［24］ Mosleh YY, Paris-Palacios S, Biagianti-Risbourg S. Metallothione-ins induction and antioxidative response in aquatic worms Tubifex tubifex（Oligochaeta, Tubificidae）exposed to copper［J］. Che-mosphere, 2006, 64（1）：121-128.
［25］Znidarsic N, Tusek-Znidaric M, Falnoga I, et al. Metallothionein-like proteins and zinc-copper interaction in the hindgut of Porcellio scaber（Crustacea：Isopoda）exposed to zinc［J］. Biol Trace Elem Res, 2005, 106（3）：253-264.
［26］黄开胜, 李思源, 何小青, 等. ICP-AES法和AAS法测定涂料中多种元素的比较研究［J］. 中国涂料, 2010, 25（2）：49-51.
［27］黄开胜, 李思源, 何小青, 等. 电感耦合等离子发射光谱（ICP-AES）和原子吸收光谱（AAS）测定涂料中多种元素的比较研究［J］. 现代涂料与涂装, 2010, 13（1）：38-40.
［28］Zhang J, Zhang M, Tian S, et al. Metallothionein 2（SaMT2）from Sedum alfredii hance confers increased Cd tolerance and accumulation in yeast and tobacco［J］. PLoS One, 2014, 9（7）：e102750.
［29］Gu CS, Liu LQ, Zhao YH, et al. Overexpression of Iris. lactea var. chinensis metallothionein llMT2a enhances cadmium tolerance in Arabidopsis thaliana［J］. Ecotox Environ Safe, 2014, 105：22-28.
［30］Tomas M, Pagani MA, Andreo CS, et al. Sunflower metallothionein family characterisation. Study of the Zn（II）- and Cd（II）-binding abilities of the HaMT1 and HaMT2 isoforms［J］. J Inorg Biochem, 2015：148：35-48.
［31］Mudalkar S, Golla R, Sengupta D, et al. Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L. , a promising biofuel plant［J］. Mol Biol Rep, 2014, 41（1）：113-124.
［32］Zhang M, Takano T, Liu S, et al. Abiotic stress response in yeast and metal-binding ability of a type 2 metallothionein-like protein（PutMT2）from Puccinellia tenuiflora［J］. Molecular Biology Reports, 2014, 41（9）：5839-5849.