TCP1: ANALYSIS OF LYSINE MODIFICATION USING PROTEIN MICROARRAYS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。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，以确定蛋白质蛋白质相互作用，小分子蛋白质相互作用，酶底物，结合和酶反应的特异性。在这里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，以确定酵母中不同的（去）乙酰化酶、甲基化酶和（去）泛素化酶的下游靶标，以产生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，并改进当前的蛋白质微阵列技术。这些研究有望为通过赖氨酸侧链上不同类型的翻译后修饰调控蛋白质功能提供一个全面的图景，并进一步提高蛋白质微阵列技术的应用。具体而言，我们计划进行以下相关活动：1）制作酵母蛋白质组芯片，并系统地测试各种赖氨酸修饰活性相关生物测定的最佳测定条件。2)目的构建包含大肠杆菌基因组中所有基因的表达文库。大肠杆菌，并纯化其编码的蛋白质，以创建一个E. coli蛋白质组微阵列;大肠杆菌蛋白质组微阵列筛选底物的新确定的E。螺旋乙酰化酶和脱乙酰化酶。3)构建人线粒体蛋白质芯片，筛选人Sirt 3 p去乙酰化酶的相互作用蛋白，并鉴定相应的乙酰转移酶。4)为了进一步提高蛋白质微阵列技术作为一种更快，更容易和用户友好的方法，可以耦合到其他检测方法，

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