TDG变体的构建、表达、纯化及其DNA修复功能研究-东南大学学报医学版

TDG变体的构建、表达、纯化及其DNA修复功能研究

单位：南京大学医药生物技术国家重点实验室,江苏南京 210093

分类号：Q786; Q754

出版年·卷·期（页码）：2012·31·第四期（389-392）

摘要：

目的:研究DNA错配修复蛋白 mTDG截短变体修复G:U错配的生物活性。方法:通过PCR扩增得到mTDG的C-端截短变体mTDG(1-279)基因片段,用Nde Ⅰ和Xho Ⅰ双酶切并回收,然后插入双酶切过的原核表达载体pET28a(+),得到pET28a-mTDG(1-279)重组质粒。将重组质粒转化表达菌株BL21(DE3),经IPTG诱导表达,过Ni⁺柱纯化得到His- mTDG(1-279)融合蛋白。最后通过TDG体外测活试验,测试mTDG(1-279)蛋白对G:U错配的修复活性。结果:mTDG(1-279)蛋白获得了表达和分离纯化,且体外测活实验表明mTDG(1-279)蛋白具有良好的G:U错配修复活性。结论:mTDG(1-279)蛋白依然具有修复G:U错配的生物活性,为进一步研究其活性结构域等结构与功能关系奠定了基础。

Objective: To investigate the G:U mismatch repair ability of truncated form of DNA repair protein mTDG. Methods: Firstly, truncated mTDG (1-279) encoding gene fragment was amplified by PCR and then digested with Nde Ⅰ and Xho Ⅰ, and inserted into the similarly digested expression vector pET28a(+). The recombinant plasmid pET28a-mTDG(1-279) was screened. The expression plasmid pET28a-mTDG(1-279) was transformed into E.coli BL21(DE3), then induced by IPTG. The recombinant protein His-mTDG(1-279) was purified through Ni⁺ affinity chromatography The repair activity of mTDG(1-279) protein towards G:U mismatch was assayed in vitro. Results: mTDG(1-279) protein was expressed and purified. The in vitro activity assay demonstrated that the truncated protein remained a good repair activity towards G:U mismatch. Conclusion: mTDG(1-279) protein has the biological activity of G:U mismatch repair. This study has laid foundation for further structure-function study, including activity domain definition.

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