TCP1: ANALYSIS OF LYSINE MODIFICATION USING PROTEIN MICROARRAYS

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 技术核心项目1 (TCP-1) 蛋白质微阵列：开发新的蛋白质微阵列技术，以识别蛋白质蛋白质相互作用、小分子蛋白质相互作用、酶底物、结合特异性和酶促反应。这里要开发的技术的一个关键新方面是使用体外翻译系统作为起点 蛋白质生产，可以显着提高产量。 我们建议研究分析赖氨酸修饰的最佳反应条件，识别酵母中不同（去）乙酰化酶、甲基化酶和（去）泛素化酶的下游靶标，创建大肠杆菌和人线粒体蛋白质微阵列以分析特定蛋白质乙酰化活性，并改进当前的蛋白质微阵列技术。这些研究有望提供通过赖氨酸侧链上不同类型的翻译后修饰调节蛋白质功能的全局图景，并进一步改进蛋白质微阵列技术的应用。具体来说，我们计划开展以下相关活动： 1) 构建酵母蛋白质组微阵列并系统测试与赖氨酸修饰活性相关的不同类型生物测定的最佳测定条件。 2) 构建包含大肠杆菌基因组所有基因的表达文库，并纯化其编码蛋白，构建大肠杆菌蛋白质组微阵列；使用这些大肠杆菌蛋白质组微阵列来筛选新鉴定的大肠杆菌的底物 乙酰酶和脱乙酰酶。 3) 构建优先的人线粒体蛋白芯片，以识别人Sirt3p脱乙酰酶的相互作用伙伴并识别相应的乙酰转移酶。 4) 进一步改进蛋白质微阵列技术，使其成为一种更快、更简单、更用户友好的方法，可以与其他检测相结合

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Technology Core Projects 1 (TCP-1) Protein microarrays: Developing new protein microarray technology to identify protein protein interactions, small moleculeprotein interactions, enzyme substrates, specificity of binding and enzymatic reactions. A key new aspect of the technology to be developed here is to use in vitro translation systems as the starting point for protein production, which can significantly increase yields. We propose to investigate the optimal reaction conditions for analyzing lysine modifications, to identify downstream targets of different (de)acetylases, methylases, and (de)ubiquitylases in yeast, to create E. coil and human mitochondrial protein microarrays for analyzing specific protein acetylation activities, and to improve current protein microarray technologies. These studies are expected to provide a global picture of regulating protein functions through different kinds of posttranslational modifications on lysine side chains, and to further improve the application of protein microarray technologies. Specifically, we plan the following interrelated activities: 1) To fabricate yeast proteome microarrays and systematically test the optimal assay conditions for different kinds of bioassays related to lysine modification activities. 2) To construct an expression library containing all genes in the genome of E. coli, and purify their encoded proteins to create an E. coli proteome microarray; use these E. coli proteome microarrays to screen for substrates of the newly identified E. coil acetylase and deacetylase. 3) To construct a prioritized human mitochondrial protein chip to identify the interacting partners of human Sirt3p deacetylase and identify the corresponding acetyltransferase(s). 4) To further improve the protein microarray technologies as a faster, easier and userfriendlier approach that can be coupled to other detection met