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

描述(申请人提供)：本申请专注于升级基于大肠杆菌的无细胞表达系统，并优化无细胞和核磁共振之间的协同作用，以筛选和评估3360个人整膜蛋白文库，用于核磁共振和X射线方法的结构研究。目前，对于膜蛋白结构的研究存在着巨大的知识鸿沟。蛋白质数据库中可用的人膜蛋白质结构不到20个。膜蛋白介导细胞与周围环境的相互作用，它们是一半商业可用药物的靶标。因此，每个新的人类膜蛋白结构都是合理结构指导药物设计的潜在靶点，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针对膜蛋白优化的现有CF表达系统，以实现正确的二硫键形成和大尺寸蛋白质的表达，并改进指定用于结晶筛选的CF表达靶标(Aim1)的纯化。我们还将利用碳纤维和核磁共振之间的协同作用来优化膜蛋白的核磁共振结构测定方法。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来结合特定部位的非天然氨基酸。我们还将使用19F和13C-甲基标记(目标2)。我们将利用这些技术改进，通过测试建议的人膜蛋白靶标的表达及其对核磁共振和X射线结构研究的适用性来全面评估它们(Aim3)。 与公共健康相关：在这项拟议的研究中，我们的目标是为膜蛋白的表达和随后的结构确定创造创新的工具，并使用这些工具来筛选一半的人类膜蛋白质组。每个人的膜蛋白结构都是合理结构指导药物设计的潜在靶点，新结构的重要性怎么强调都不为过。为了达到所提出的目标，我们将修改和升级一种新的体外表达系统，以生产人的完整膜蛋白，并将其与高分辨率核磁共振技术相结合。

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.