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: 8517748
• 负责人: SENYON CHOE
• 金额: $ 28.66万
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517748
• 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; 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; screening; tool; vector

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.

描述（由申请人提供）：本申请重点是升级基于大肠杆菌的无细胞表达系统，并优化无细胞和 NMR 之间的协同作用，以通过 NMR 和 X 射线方法筛选和评估 3360 个人类整合膜蛋白库的结构研究。目前，膜蛋白结构存在巨大的知识缺口。蛋白质数据库中提供的人类膜蛋白结构不到 20 个。膜蛋白介导细胞与周围环境的相互作用，它们是一半市售药物的目标。因此，每个新的人类膜蛋白结构都是合理结构引导药物设计的潜在目标，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针对膜蛋白优化的现有 CF 表达系统，以实现适当的二硫键形成和大尺寸蛋白表达，并改进指定用于结晶筛选 (Aim1) 的 CF 表达靶标的纯化。我们还将利用 CF 和 NMR 之间的协同作用，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 来掺入位点特异性非天然氨基酸。我们还将采用 19F 和 13C-甲基标记（目标 2）。我们将利用这些技术改进，通过测试其表达及其对 NMR 和 X 射线结构研究 (Aim3) 的适用性，全面评估所提出的人膜蛋白靶标。 公共健康相关性：在这项拟议的研究中，我们的目标是创建用于膜蛋白表达和随后结构测定的创新工具，并使用这些工具来筛选一半的人类膜蛋白质组。每个人类膜蛋白结构都是合理结构引导药物设计的潜在目标，新结构的重要性怎么强调都不为过。为了实现拟议的目标，我们将修改和升级一种新型体外表达系统，以产生人类整合膜蛋白，并将其与高分辨率核磁共振技术相结合。

项目成果

Drug screening strategy for human membrane proteins: from NMR protein backbone structure to in silica- and NMR-screened hits.

• DOI: 10.1016/j.bbrc.2014.01.179
• 发表时间: 2014-03-21
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Lindert, Steffen;Maslennikov, Innokentiy;Chiu, Ellis J. C.;Pierce, Levi C.;McCammon, J. Andrew;Choe, Senyon
• 通讯作者: Choe, Senyon

Advances in NMR structures of integral membrane proteins.

• DOI: 10.1016/j.sbi.2013.05.002
• 发表时间: 2013-08
• 期刊: Current opinion in structural biology
• 影响因子: 6.8
• 作者: Maslennikov I;Choe S
• 通讯作者: Choe S