Pirzadeh Soheil, Shahpiri Azar
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran.
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran.
Int J Biol Macromol. 2016 Jul;88:491-6. doi: 10.1016/j.ijbiomac.2016.04.021. Epub 2016 Apr 11.
Metallothioneins (MTs) are a family of Cys-rich, low molecular weight, cytoplasmic metal binding proteins. MTs are present in all eukaryotes as well as some prokaryotes. Plant MTs are divided into four types based on Cys distribution pattern in their amino acid sequences. In the present work, the gene encoding OsMTI-2b, a type 2 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into Escherichia coli strain Rosetta (DE3). Following the induction with Isopropyl β-d-1-thiogalactopyranoside the OsMTI-2b was expressed as carboxyl-terminal extensions of glutathione-S-transferase (GST-tag), a 6His-tag, and an S-tag. The expressed recombinant fusion protein was named GST-OsMTI-2b. As compared with control, transgenic E. coli cells expressing GST-OsMTI-2b accumulated more Pb(2+), Ni(2+), Cd(2+), Zn(2+) and Cu(2+) from culture medium and showed increased tolerance against these metals. Furthermore the E. coli cells expressing OsMTI-2b accumulated significantly higher Pb(2+) than previously made strains which expressing other rice OsMT isoforms. The recombinant GST-OsMTI-2b was purified using affinity chromatography. According to in vitro assays the protein GST-OsMTI-2b was able to form complexes with Pb(2+), Ni(2+), Cd(2+) and Zn(2+). However, the binding ability for the different metals differed in the order: Pb(2+)>Cd(2+)>Zn(2+)>Ni(2+).
金属硫蛋白（MTs）是一类富含半胱氨酸、低分子量的细胞质金属结合蛋白家族。MTs存在于所有真核生物以及一些原核生物中。根据植物MTs氨基酸序列中的半胱氨酸分布模式，可将其分为四种类型。在本研究中，将编码水稻中发现的2型MT即OsMTI-2b的基因克隆到pET41a载体中。将得到的构建体转化到大肠杆菌Rosetta (DE3)菌株中。用异丙基-β-D-硫代半乳糖苷诱导后，OsMTI-2b作为谷胱甘肽-S-转移酶（GST标签）、6His标签和S标签的羧基末端延伸物表达。表达的重组融合蛋白命名为GST-OsMTI-2b。与对照相比，表达GST-OsMTI-2b的转基因大肠杆菌细胞从培养基中积累了更多的Pb(2+)、Ni(2+)、Cd(2+)、Zn(2+)和Cu(2+)，并对这些金属表现出更高的耐受性。此外，表达OsMTI-2b的大肠杆菌细胞积累的Pb(2+)明显高于先前构建的表达其他水稻OsMT同工型的菌株。重组GST-OsMTI-2b通过亲和层析进行纯化。根据体外试验，蛋白GST-OsMTI-2b能够与Pb(2+)、Ni(2+)、Cd(2+)和Zn(2+)形成复合物。然而，对不同金属的结合能力顺序为：Pb(2+)>Cd(2+)>Zn(2+)>Ni(2+)。
