Cell-free expression of human integral membrane proteins for structural studies

描述（由申请人提供）：本申请的重点是升级基于大肠杆菌的无细胞表达系统，优化无细胞和核磁共振之间的协同作用，通过核磁共振和x射线方法筛选和评估3360个人类整体膜蛋白库的结构研究。目前，关于膜蛋白结构的知识还存在着巨大的空白。蛋白质数据库中只有不到20种人膜蛋白结构。膜蛋白介导细胞与周围环境的相互作用，它们是半数市售药物的靶标。因此，每一种新的人膜蛋白结构都是合理结构指导药物设计的潜在靶标，新结构的重要性怎么强调都不为过。我们建议改进我们实验室优化的现有的膜蛋白CF表达系统，使其能够形成适当的二硫桥和大尺寸的蛋白表达，并改进用于结晶筛选的CF表达靶点（Aim1）的纯化。我们还将利用CF和NMR的协同作用，优化膜蛋白的NMR结构测定方法。我们将改进我们实验室开发的组合双同位素标记（CDL）策略，并使用CF结合位点特异性非天然氨基酸。我们还将使用19F和13c -甲基标记（Aim 2）。我们将利用这些技术改进，通过测试其表达及其在核磁共振和X射线结构研究（Aim3）中的适用性，全面评估拟议的人膜蛋白靶点。

DESCRIPTION (provided by applicant): This application focuses on upgrading the E. coli-based cell-free expression system and on optimizing the synergy between cell-free and NMR to screen and evaluate for structural studies by NMR and X-ray methods a library of 3360 human integral membrane proteins. Currently there exists an enormous knowledge gap for membrane protein structures. Fewer than 20 human membrane protein structures are available in the Protein Data Bank. Membrane proteins mediate cellular interactions with the surrounding, and they are targeted by half of the commercially available drugs. Each new human membrane protein structure is, therefore, a potential target of rational structure-guided drug design, and the significance of new structures cannot be overstated. We propose to improve the existing CF expression system optimized in our lab for membrane proteins to enable proper disulfide bridge formation and a large-size protein expression, and to improve purification of CF-expressed targets designated for crystallization screening (Aim1). We will also optimize the NMR structure determination method for membrane proteins utilizing the synergy between CF and NMR. We will improve the combinatorial dual-isotope labeling (CDL) strategy developed in our lab and use CF to incorporate site-specific unnatural amino acids. We will also employ 19F and 13C-methyl labeling (Aim 2). We will use these technical improvements to comprehensively evaluate the proposed human membrane protein targets by testing their expression and their suitability for NMR and X- ray structural studies (Aim3). PUBLIC HEALTH RELEVANCE: In this proposed study we aim to create innovative tools for expression and subsequent structure determination of membrane proteins and use these tools to screen half of the human membrane proteome. Each human membrane protein structure is a potential target for rational structure-guided drug design and the significance of new structures cannot be overstated. To achieve proposed aims we will modify and upgrade a novel in vitro expression system to produce human integral membrane proteins and combine it with high resolution NMR techniques.