拟南芥钙依赖性核酸酶的分子克隆、表达及特性分析
Molecular cloning, expression, and characterization of a Ca2+-dependent nuclease of Arabidopsis thaliana.
作者信息
Guo Kunyuan, Liu Shenkui, Takano Tetsuo, Zhang Xinxin
机构信息
Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, China.
出版信息
Protein Expr Purif. 2012 May;83(1):70-4. doi: 10.1016/j.pep.2012.03.007. Epub 2012 Mar 17.
A gene encoding a Ca(2+)-dependent nuclease (AtCaN2) consisting of 226 amino acids from Arabidopsis thaliana was isolated. The AtCaN2 gene was expressed in Escherichia coli as a His fusion protein. The His-AtCaN2 fusion protein was purified by affinity chromatography using a Ni-NTA column. Western blot analysis using an anti-His antibody showed that the His-AtCaN2 fusion protein was not degraded. The nuclease activity of the His-AtCaN2 fusion protein was highest at 37°C, required a neutral or weakly-alkaline environment, and was stimulated by Ca(2+) and Mg(2+), but inhibited by Zn(2+) and high concentration of Mn(2+).
从拟南芥中分离出一个编码由226个氨基酸组成的钙依赖性核酸酶（AtCaN2）的基因。AtCaN2基因在大肠杆菌中作为His融合蛋白表达。His-AtCaN2融合蛋白通过使用镍-氮川三乙酸（Ni-NTA）柱的亲和层析法进行纯化。使用抗His抗体的蛋白质免疫印迹分析表明His-AtCaN2融合蛋白未被降解。His-AtCaN2融合蛋白的核酸酶活性在37°C时最高，需要中性或弱碱性环境，并且受到Ca(2+)和Mg(2+)的刺激，但受到Zn(2+)和高浓度Mn(2+)的抑制。
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