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

总结 该应用程序的重点是升级E。基于大肠杆菌的无细胞表达系统和基于 优化无细胞和NMR之间的协同作用，以筛选和评估结构研究 通过NMR和X-射线方法，获得了3360个人完整膜蛋白的文库。目前 对于膜蛋白结构存在巨大的知识缺口。不到20 人膜蛋白结构可在蛋白质数据库中获得。膜蛋白 介导细胞与周围环境的相互作用，它们是一半的细胞的目标。 市售药物。因此，每一种新的人类膜蛋白结构都是 合理结构导向药物设计的潜在目标，以及新结构的意义 怎么说都不过分我们建议改进现有的CF表达系统， 我们实验室的膜蛋白，使适当的二硫键形成和大尺寸的 蛋白质表达，并改善指定用于治疗癌症的CF表达靶标的纯化。 结晶筛选（Aim 1）。我们还将优化核磁共振结构测定方法 膜蛋白利用CF和NMR之间的协同作用。我们会完善 组合双同位素标记（CDL）策略，并使用CF 掺入位点特异性非天然氨基酸。我们还将使用19 F和13 C-甲基标记 (Aim 2）。我们将利用这些技术改进来全面评估拟议的 通过测试它们的表达和它们对NMR和X-射线衍射的适用性， 射线结构研究（Aim 3）。

Summary 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).