Saffar B, Mehri Ghahfarrokhi A, Mahnam K, Mobini-Dehkordi M
a Faculty of Science, Department of Genetics , Shahrekord University , Shahrekord , Iran.
b Biotechnology Research Institute , Shahrekord University , Shahrekord , Iran.
J Biomol Struct Dyn. 2015;33(11):2347-59. doi: 10.1080/07391102.2015.1054431. Epub 2015 Jun 19.
The improved Cd(2+) surface affinity characteristics of a mutated cyanobacterial metallothionein SmtA (K45C) were investigated via experimental and theoretical methods. Molecular dynamics simulations were carried out using a model of Cd(2+) and other ions enclosed in a fully hydrated simulation box with the wild-type or mutated SmtA protein. The theoretical results suggested that mutated SmtA was more powerful in absorption of Cd(2+) than the wild-type protein. Then, the mutated smtA gene (from Synechococcus PCC 7942) was synthesized by simplified gene synthesis method and expressed on isopropyl-beta-d-thiogalactopyranoside induction. The protein expression was investigated by SDS-PAGE and verified by Western blotting. Finally, cadmium uptake ratio of mutant protein toward wild type was analyzed by atomic absorption. This study is the first example of cytoplasmic expression of a mutant protein. Experimental results also verified that the mutation intensifies uptake of Cd(2+) ions.
通过实验和理论方法研究了突变型蓝藻金属硫蛋白SmtA（K45C）改善的Cd(2+)表面亲和特性。使用包含野生型或突变型SmtA蛋白的完全水合模拟盒中的Cd(2+)和其他离子模型进行分子动力学模拟。理论结果表明，突变型SmtA对Cd(2+)的吸收能力比野生型蛋白更强。然后，通过简化基因合成方法合成了突变型smtA基因（来自聚球藻PCC 7942），并在异丙基-β-D-硫代半乳糖苷诱导下表达。通过SDS-PAGE研究蛋白质表达，并通过蛋白质免疫印迹法进行验证。最后，通过原子吸收分析突变蛋白对野生型的镉摄取率。本研究是突变蛋白在细胞质中表达的首个实例。实验结果也证实了该突变增强了对Cd(2+)离子的摄取。
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