|
|||||
|
|
| DNA错配修复蛋白MutS和MutL的相互作用研究 | |
| 引用本文： | 毕利军,张先恩,周亚凤,张治平.DNA错配修复蛋白MutS和MutL的相互作用研究[J].生物化学与生物物理进展,2005,32(12):1178-1184. |
| 作者姓名： | 毕利军 张先恩 周亚凤 张治平 |
| 作者单位： | 1. 中国科学院生物物理研究所,生物大分子国家重点实验室,北京,100101 2. 中国科学院生物物理研究所,生物大分子国家重点实验室,北京,100101;中国科学院武汉病毒研究所,病毒学国家重点实验室,武汉,430071 3. 中国科学院武汉病毒研究所,病毒学国家重点实验室,武汉,430071 |
| 基金项目： | 国家自然科学基金资助项目(30500097), 国家“九五”攻关项目和中国科学院“九五”重大项目(KSCX1-06-01) |
| 摘 要： | MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白，并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL)，MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析，结果表明有与预期分子质量相应的诱导表达条带出现，其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白，其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台. |
| 关 键 词： | 融合蛋白，绿色荧光蛋白，Strep tagⅡ，MutS，MutL，相互作用 |
| 收稿时间： | 06 13 2005 12:00AM |
| 修稿时间： | 2005-06-132005-08-28 |
Observation of The Interaction Between MutS and MutL Mismatch Repair Proteins by Fusion Protein Systems |
|
| BI Li-Jun,ZHANG Xian-En,ZHOU Ya-Feng and ZHANG Zhi-Ping.Observation of The Interaction Between MutS and MutL Mismatch Repair Proteins by Fusion Protein Systems[J].Progress In Biochemistry and Biophysics,2005,32(12):1178-1184. | |
| Authors: | BI Li-Jun ZHANG Xian-En ZHOU Ya-Feng and ZHANG Zhi-Ping |
| Institution: | IBP-WIV Joint Research Group for Analytical Biotechnology, National Laboratory of Biomacromolecules, Institute of Biophysics and National Laboratory of Viology, Wuhan Institute of Virology, The Chinese Academy of Sciences, Beijing 100101, China;IBP-WIV Joint Research Group for Analytical Biotechnology, National Laboratory of Biomacromolecules, Institute of Biophysics and National Laboratory of Viology, Wuhan Institute of Virology, The Chinese Academy of Sciences, Beijing 100101, China;IBP-WIV Joint Research Group for Analytical Biotechnology, National Laboratory of Biomacromolecules, Institute of Biophysics and National Laboratory of Viology, Wuhan Institute of Virology, The Chinese Academy of Sciences, Beijing 100101, China;IBP-WIV Joint Research Group for Analytical Biotechnology, National Laboratory of Biomacromolecules, Institute of Biophysics and National Laboratory of Viology, Wuhan Institute of Virology, The Chinese Academy of Sciences, Beijing 100101, China |
| Abstract: | MutL and MutS or their homologues are two crucial proteins of DNA mismatch repair (MMR) system. A new method was described for observation of the interaction between MutS and MutL which is based on the fusion gene/fusion protein technique. Three fusion proteins, MutL-GFP fusion (Trx-His6-GFP-(Ser-Gly)6-MutL), MutL-Strep tag Ⅱ fusion (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) and MutS fusion (Trx-His6-(Ser-Gly)6-MutS), were constructed and expressed in E. coli AD494 (DE3). Interaction assay between MutS and MutL was performed in a 96-well microtiter plate.MutS fusion protein was immobilized on the wells and provided a surface for the interaction between MutS and MutL.Results showed that only after binding of MutS to the mismatched DNA, there was an interaction between MutS and MutL.The binding events could be indicated by GFP signal or the signal generated from alkaline phosphatase and its substrate. In addition, the method based on fusion molecular system also serve as a model for studies on the interactions among other proteins or biomolecules. |
| Keywords: | fusion protein GFP strep tagⅡ MutS MutL interaction |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ | |
| 点击此处可从《生物化学与生物物理进展》浏览原始摘要信息 | |
| 点击此处可从《生物化学与生物物理进展》下载免费的PDF全文 | |