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

用于结构研究的人类整合膜蛋白的无细胞表达

• 批准号: 8312574
• 负责人: SENYON CHOE
• 金额: $ 29.7万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312574
• 关键词: Amino Acid Sequence; Amino Acids; Cell physiology; Cell-Free System; Cells; Crystallization; Databases; Detection; Disulfides; Drug Design; Escherichia coli; Goals; Human; In Vitro; Integral Membrane Protein; Isotope Labeling; Knowledge; Label; Libraries; Mediating; Membrane; Membrane Proteins; Methods; Peptide Sequence Determination; Pharmaceutical Preparations; Process; Proteins; Proteome; Resolution; Roentgen Rays; Sampling; Screening procedure; Signal Transduction; Site; Spectrum Analysis; Speed; Structure; System; Techniques; Testing; base; combinatorial; cysteine rich protein; disulfide bond; improved; innovation; novel; overexpression; process optimization; protein expression; protein structure; public health relevance; tool; vector

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).

摘要 这项应用的重点是升级基于大肠杆菌的无细胞表达系统，以及 优化无细胞核磁共振之间的协同作用以筛选和评估结构研究 通过核磁共振和X-射线分析方法，构建了3360个人完整膜蛋白文库。目前 膜蛋白结构的研究存在着巨大的知识鸿沟。少于20个 蛋白质数据库中提供了人膜蛋白质结构。膜蛋白 调节细胞与周围环境的相互作用，它们是一半的 市面上可以买到的药品。因此，每个新的人类膜蛋白结构都是一种 合理结构导向药物设计的潜在目标和新结构的意义 怎么夸大都不为过。我们建议改进现有的优化的CF表达系统 我们实验室的膜蛋白能够使适当的二硫键形成和大尺寸 蛋白表达，并改进指定的Cf表达靶点的纯化 结晶筛选(Aim1)。我们还将优化核磁共振结构测定方法 用于利用CF和核磁共振之间的协同作用的膜蛋白质。我们将改善 我们实验室开发的组合双同位素标记(CDL)策略，并使用CF 加入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来综合评估拟议的 检测人膜蛋白靶标的表达及其对核磁共振和X-射线分析的适用性 射线结构研究(Aim3)。