Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran.
Department of Agricultural Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran.
Int J Biol Macromol. 2015 Feb;73:154-9. doi: 10.1016/j.ijbiomac.2014.10.067. Epub 2014 Nov 28.
Metallothioneins (MTs) are low-molecular weight proteins with high Cys content and a high affinity for metals. Plant MTs are classified into four types based on the arrangement of Cys in their amino acid sequences. In the present study, the gene encoding OsMTI-3a, a type 3 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl β-D-1-thiogalactopyranoside, the OsMTI-3a was expressed as glutathione-S-transferase (GST)-tagged fusion protein. In comparison to control strain, the cells expressing GST-OsMTI-3a accumulated more Cd(2+), Ni(2+) and Zn(2+) when they were grown in the medium containing CdCl2, NiCl2 or ZnSO4. The recombinant GST-OsMTI-3a was purified using affinity chromatography. The UV absorption spectra recorded after the reconstitution of the apo-protein with different metals confirmed that GST-OsMTI-3a was able to form complexes with Cd(2+), Ni(2+), and Zn(2+). The reaction of the protein-metal complexes with 5-5-dithiobis (2-nitrobenzoic) revealed that the order of affinity of GST-OsMTI-3a toward different metals was Ni(2+)≥Cd(2+)>Zn(2+)>Cu(2+).
金属硫蛋白（MTs）是一种富含半胱氨酸且对金属具有高亲和力的低分子量蛋白。植物 MT 可根据其氨基酸序列中半胱氨酸的排列方式分为 4 种类型。本研究克隆了水稻中发现的第 3 型 MT 基因 OsMTI-3a，并将其插入 pET41a 载体。所得构建体转化入大肠杆菌 Rosetta（DE3）菌株。异丙基-β-D-1-硫代半乳糖苷诱导后，OsMTI-3a 作为谷胱甘肽-S-转移酶（GST）标记融合蛋白表达。与对照菌株相比，在含有 CdCl2、NiCl2 或 ZnSO4 的培养基中生长时，表达 GST-OsMTI-3a 的细胞积累了更多的 Cd（2+）、Ni（2+）和 Zn（2+）。使用亲和层析纯化重组 GST-OsMTI-3a。用不同金属重新构建脱辅基蛋白后记录的紫外吸收光谱证实，GST-OsMTI-3a 能够与 Cd（2+）、Ni（2+）和 Zn（2+）形成复合物。蛋白-金属复合物与 5,5-二硫代双（2-硝基苯甲酸）的反应表明，GST-OsMTI-3a 对不同金属的亲和力顺序为 Ni（2+）≥Cd（2+）>Zn（2+）>Cu（2+）。
