体内以DNA为中心的互作蛋白的鉴定与定量方法学研究

项目名称： 体内以DNA为中心的互作蛋白的鉴定与定量方法学研究

项目编号： No.31300041

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 李庆刚

作者单位： 中国科学院天津工业生物技术研究所

项目金额： 22万元

中文摘要： DNA与蛋白质因子的相互作用是DNA复制与修复、基因转录调控的重要基础。目前已经发展的体内体外DNA与蛋白质互作研究方法不能有效地富集鉴定与给定DNA相互作用的蛋白质，难以了解参与调控的全部蛋白组分和动态调控机制。针对这一问题，本项目设计了一种特殊工具质粒，携带亲和纯化和蛋白质相对定量标签，结合高分辨质谱技术，可针对任意给定目标DNA序列，鉴定体内与其发生相互作用的蛋白质分子并实现相对定量。本研究以转录调控机理较清楚的大肠杆菌二氨基庚二酸脱羧酶lysA基因启动子为例对方法进行优化。最终目标是突破以往转录调控研究方法的局限，鉴定体内生物环境下与任意给定DNA区域发生相互作用的蛋白质因子单体、蛋白质复合体所有成分，定量分析给定DNA区域与每种蛋白的结合比率，从而全面解析给定DNA序列的动态调控行为，为深入理解生命体生理调控机制奠定基础。

中文关键词： DNA-蛋白相互作用；转录调控；细胞对小分子响应；工业菌株提升；

英文摘要： DNA-protein interaction plays a key role in the regulation of cellular functions such as DNA replication and reparation, gene transcription, and etc. All the existing methods such as chromatin immunoprecipitation (CHIP), DNA affinity chromatography, electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), yeast one-hybrid assay have limitations to identify all the components involved in the in vivo interactions with a given DNA region and quantify the in vivo interaction dynamics simultaneously. In this study, we design a powerful plasmid, harboring protein tag for affinitive purification and relative quantification. In combination with high-end mass spectrometry technology, our method can identify and quantify all the proteins which interact with the given DNA fragment in vivo. We take the well-known diaminopimelate decarboxylase gene promoter as example to evaluate and optimize our method. The final goal of this method is to break down the limitation of the existing methods, provide a scalable tools to identify all the components of the proteins interacting with any certain DNA region in vivo, and at mean while provide the key information such as occupancy ratio of the proteins on the DNA region. The successful establishment of this method will largely strengthen the global understanding o

英文关键词： DNA-protein interaction；transcriptional regulation；cellular response to small molecular；industrial strain improvement；