TCP1: ANALYSIS OF LYSINE MODIFICATION USING PROTEIN MICROARRAYS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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，用于鉴定蛋白质相互作用、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蛋白质生产的起点，这可以显着提高产量。我们建议研究分析赖氨酸修饰的最佳反应条件，确定酵母中不同（去）乙酰化酶、甲基化酶和（去）泛素化酶的下游靶点，建立E. coil和人类线粒体蛋白质微阵列，用于分析特定蛋白质乙酰化活性，并改进现有的蛋白质微阵列技术。这些研究有望为通过不同类型的赖氨酸侧链翻译后修饰调控蛋白质功能提供一个全局图景，并进一步完善蛋白质微阵列技术的应用。具体而言，我们计划以下相关活动：1)制作酵母蛋白质组微阵列，并系统地测试与赖氨酸修饰活性相关的各种生物测定的最佳测定条件。2)构建包含大肠杆菌基因组中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芯片；利用这些大肠杆菌蛋白质组芯片筛选新鉴定的大肠杆菌乙酰化酶和去乙酰化酶的底物。3)构建优先级人线粒体蛋白芯片，鉴定人Sirt3p去乙酰化酶的相互作用伙伴，鉴定相应的乙酰转移酶。4)进一步改进蛋白质微阵列技术，使其成为一种更快、更容易、更友好的方法，可以与其他检测方法相结合

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