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A large-scale analysis of protein-protein interactions using Escherichia coli proteome chips

使用大肠杆菌蛋白质组芯片大规模分析蛋白质-蛋白质相互作用

基本信息

批准号：
17201042
负责人：
DOI Nobuhide
金额：
$ 22.71万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Although protein chips are powerful tools for high-throughput analysis of protein-protein interactions (PPIs), a large-scale PPI analysis using proteome chips has not been reported yet. Here we report the construction and application of Escherichia coli proteome chips for detection of PPIs.First we optimized the condition of PPI analysis using model E. coli proteins with regard to immobilization of proteins expressed in E. coli and detection with fluorescently labeled proteins synthesized in a cell-free system. We prepared proteome chips containing 4337 E. coli open reading frames (ORFs) and successfully detected known PPIs.Next we attempted to perform high-throughput fluorescence labeling of various proteins in a multi-well format. We prepared template DNA of 162 E. colt genes by PCR and synthesized fluorescently labeled proteins with the PURE E. coil reconstituted system containing Cy3-dC-puromycin in 96-well microplates. One hundred and forty-five out of 162 full- length proteins (-90%) were successfully labeled with Cy3.Finally we performed a large-scale analysis of E. colt PPIs. Overexpressed products from 4337 ORFs of E. call were printed on-100 slides in duplicate or quadruplicate, and each slide was probed by the Cy3-labeled proteins. As a result, total of 1014 PPIs including novel interactions were detected at high signal-noise ratios. When arbitrary chosen PPIs were tested by pull-down assays,-50% of PPIs were verified. These results indicate that the method established in this study should be useful to study the large-scale analysis of bacterial PPI networks.

虽然蛋白质芯片是高通量分析蛋白质-蛋白质相互作用（PPI）的有力工具，但使用蛋白质组芯片进行大规模PPI分析的报道还没有。本文报道了用于PPI检测的大肠杆菌蛋白质组芯片的构建和应用。coli蛋白质的固定化。大肠杆菌和检测与荧光标记的蛋白质合成的无细胞系统。我们制备了含有4337 E的蛋白质组芯片。coli开放阅读框（ORF），成功检测到已知的PPI。制备了162 E的模板DNA。colt基因，用PURE E.在96孔微孔板中的含有Cy 3-dC-嘌呤霉素的螺旋重构系统。在162个全长蛋白质中，有145个（约90%）被Cy 3成功标记。小马PPI。从E.一式两份或一式四份在~ 100个载玻片上印刷，每个载玻片用Cy 3标记的蛋白质探测。结果，在高信噪比下共检测到1014种PPI，包括新型相互作用。当通过下拉试验检测任意选择的PPI时，约50%的PPI得到验证。这些结果表明，在这项研究中建立的方法应该是有用的，研究大规模的细菌PPI网络的分析。