Membrane Protein Expression Center

膜蛋白表达中心

• 批准号: 8146423
• 负责人: Robert M Stroud
• 金额: $ 9.41万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-24 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8146423
• 关键词: Algorithms; Area; Awareness; Back; Bacteriophages; Bioenergetics; Biological Assay; Biological Testing; Biology; Carrier Proteins; Caustics; Cells; Cloning; Cloning Vectors; Collaborations; Complex; Crystallization; Crystallography; Detergents; Development; Development Plans; Diffusion; Environment; Epitopes; Evolution; Fab Immunoglobulins; Fission Yeast; Future; G-Protein-Coupled Receptors; Genes; Goals; Health; Heterogeneity; Homologous Gene; Human; Human Resources; Immunoprecipitation; In Vitro; Ion Channel; Ligands; Ligase; Light; Lipids; Membrane; Membrane Proteins; Methodology; Methods; Micelles; Microfluidics; Miniaturization; Molecular Chaperones; Mutate; Nuclear Pore; Organism; Pharmaceutical Preparations; Phase; Pichia; Plasmodium falciparum; Porifera; Post-Translational Protein Processing; Principal Investigator; Prokaryotic Cells; Protein Binding; Proteins; Proteome; Pseudomonas aeruginosa; Research Personnel; Robotics; Roentgen Rays; Saccharomyces cerevisiae; Scanning; Screening procedure; Sensory Receptors; Source; Staphylococcus aureus; Structure; System; Testing; Therapeutic; Time; Variant; Vision; Visit; Yeasts; base; design; detector; expectation; experience; genome-wide; high throughput screening; improved; in vivo; novel; pathogen; programs; protein complex; protein expression; protein purification; protein structure; protein transport; public health relevance; receptor; response; vapor; web site

DESCRIPTION (provided by applicant): This is a renewal application for the Roadmap Initiative (RFA-RM-08-019) 'Membrane Protein Expression Center' (MPEC). The goals are to develop, apply, and disseminate novel expression, purification and crystallization of membrane proteins including hetero-oligomeric membrane protein complexes, targeted toward their structure determination as a means of delineating ligand or protein binding, and function. The focus is toward human health, hence on methods appropriate for eukaryotic membrane proteins that generally require eukaryotic expression systems, alongside the relatively simpler bacterial expression of prokaryotic membrane proteins appropriate sometimes to homologs. New initiatives currently in development are: (1) principles of gene redesign to optimize expression. (2) a new cell free expression method from yeast, a eukaryotic source. (3) cell free synthesis in the context of lipids that can proceed directly to mesophase crystallization. (4) presentation of membrane proteins for optimization of Fab fragments as crystallization chaperones. (5) means of expressing partners and assembling functional membrane protein heterooligomeric complexes in a robust yeast expression system. (6) expression in a human HEKs cells mutated to minimize heterogeneity in post translational modification. (7) identifying partners in functional hetero-oligomeric complexes of 500 yeast membrane proteins (8) membrane protein purification. (9) microfluidic applications of lipidic sponge phase crystallization, and crystallization in microfluidic chips that can be directly inserted into the X-ray beam for screening by X-rays. (10) Target selection based on value to human health. The expression systems harness a suite of Ligase Independent Cloning (LIC) vectors developed at MPEC for insertion of genes into specially adapted forms of S. cerevisiae, Pichia pastoris, or HEKs 293 cells. The focus toward human health is reflected in the choice of human, pathogen, occasionally prokaryotic homologs, and other high value targets supported by Principal Investigators and collaborators with experience in the structural and functional biology of particular membrane protein classes. Thus identifying the most important targets and providing vision and focus for high impact in each area. These areas include channels, ion channels, transporters, secondary transporters, proteins of bioenergetic importance, G Protein Coupled Receptors, neuroreceptors, nuclear pores, intracellular receptors including that for the unfolded protein response. PUBLIC HEALTH RELEVANCE: Membrane proteins constitute ~30% of all eukaryotic proteins and are targets for over 40% of all drugs in use today, however only in the past year have structural interactions of the first eukaryotic membrane protein with a drug emerged. This proposal seeks to overcome roadblocks to expression, purification and structural determination of membrane proteins in general, and targets of importance to human health in particular. Functional assays will serve to assist in discovery of therapeutics and understanding function.

描述（由申请人提供）：这是路线图倡议 (RFA-RM-08-019)“膜蛋白表达中心”(MPEC) 的续展申请。目标是开发、应用和传播膜蛋白（包括异源寡聚膜蛋白复合物）的新型表达、纯化和结晶，针对其结构确定作为描绘配体或蛋白质结合和功能的手段。重点是人类健康，因此适合通常需要真核表达系统的真核膜蛋白的方法，以及有时适合同源物的原核膜蛋白的相对简单的细菌表达。目前正在开发的新举措有：（1）基因重新设计原则以优化表达。 (2)一种来自真核来源酵母的新的无细胞表达方法。 (3)在脂质的情况下进行无细胞合成，可以直接进行中间相结晶。 (4) 膜蛋白的呈现，用于优化作为结晶伴侣的 Fab 片段。 (5)在稳健的酵母表达系统中表达配偶体和组装功能性膜蛋白异寡聚复合物的方法。 (6)人类HEKs细胞中的表达发生突变，以尽量减少翻译后修饰中的异质性。 (7) 鉴定 500 种酵母膜蛋白的功能性异源寡聚复合物中的伙伴 (8) 膜蛋白纯化。 (9)脂质海绵相结晶的微流控应用，以及可直接插入X射线束中进行X射线筛查的微流控芯片中的结晶。 (10)基于对人类健康价值的目标选择。该表达系统利用 MPEC 开发的一套连接酶独立克隆 (LIC) 载体，将基因插入专门适应形式的酿酒酵母、巴斯德毕赤酵母或 HEKs 293 细胞中。对人类健康的关注体现在对人类、病原体、偶尔的原核同源物和其他高价值靶标的选择上，这些靶标由在特定膜蛋白类别的结构和功能生物学方面拥有丰富经验的主要研究者和合作者支持。从而确定最重要的目标，并为每个领域的高影响力提供愿景和重点。这些区域包括通道、离子通道、转运蛋白、二级转运蛋白、具有生物能量重要性的蛋白质、G蛋白偶联受体、神经受体、核孔、细胞内受体，包括用于未折叠蛋白质反应的受体。公共健康相关性：膜蛋白约占所有真核蛋白的 30%，并且是当今使用的所有药物中超过 40% 的靶标，然而直到去年才出现第一个真核膜蛋白与药物的结构相互作用。该提案旨在克服膜蛋白表达、纯化和结构测定的障碍，特别是对人类健康重要的目标。功能测定将有助于发现治疗方法和理解功能。