title: Functional characterization of a type 2 metallothionein isoform (OsMTI-2b) from rice.
abstract: 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+).
authors.affiliation: Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran.
authors.forename: Soheil
authors.initials: S
authors.lastname: Pirzadeh
authors.affiliation: Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran. Electronic address: a.shahpiri@cc.iut.ac.ir.
authors.forename: Azar
authors.initials: A
authors.lastname: Shahpiri
mesh_terms: D002104:Cadmium / Q000633:toxicity; D003300:Copper / Q000633:toxicity; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D007854:Lead / Q000633:toxicity; D008668:Metallothionein / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009532:Nickel / Q000633:toxicity; D012275:Oryza / Q000737:chemistry; D020033:Protein Isoforms / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015032:Zinc / Q000633:toxicity
chemical_list: D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D007854:Lead; D003300:Copper; D009532:Nickel; D008668:Metallothionein; D015032:Zinc
keywords: Heavy metal tolerance; Metal-binding ability; Metallothionein; Rice; Type 2 MT