Innovative Methods for Membrane Protein Crystallization

膜蛋白结晶的创新方法

基本信息

批准号：
7880319
负责人：
Lawrence J Delucas
金额：
$ 12.62万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-17 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7880319
关键词：
Accounting Active Sites Address Adopted Amino Acids Antibodies Area Asthma Biological Buffers Carbohydrates Cardiovascular system Case Mixes Categories Cell Cycle Cell Death Cell Line Cells Central Nervous System Diseases Chemicals Chromatography Classification Collaborations Communities Complex Crystal Formation Crystallization Crystallography Cystic Fibrosis Detergents Development Diagnostic Drug Design Engineering Enzymes Escherichia coli Excision Funding Future G-Protein-Coupled Receptors Gene Delivery Gene Expression Generations Genomics Homologous Protein Inclusion Bodies Individual Infection Inflammation Insecta Integral Membrane Protein Ion Channel Knowledge Lead Left Lipids Liquid substance Malignant Neoplasms Mammalian Cell Maps Marketing Membrane Membrane Biology Membrane Proteins Metabolic Methods Minor Modeling Molecular Neuraxis Obesity Pain Play Point Mutation Post-Translational Protein Processing Precipitation Principal Investigator Process Production Proteins Proteolysis Receptor Signaling Recombinant Proteins Recombinants Relative (related person)Research Research Personnel Robotics Role Saccharomyces Saccharomyces cerevisiae Scientist Screening procedure Semliki forest virus Solubility Solutions Solvents Source Specialized Center Structure Surface Suspension Culture System Technology Temperature Testing Therapeutic Time Toxic effect Treatment Protocols Vaccinia virus Viral Virus Work aqueous base cell growth cofactor flexibility gastrointestinal glycosylation improved innovation innovative technologies knowledge base macromolecule milligram novel diagnostics novel strategies novel therapeutics overexpression prevent programs protein expression protein folding protein structure receptor research study stable cell line structural biology structural genomics success technology development vector

项目摘要

DESCRIPTION (provided by applicant): Project Summary: This project combines innovative technologies in eukaryotic membrane protein expression, solubilization, stablization and crystallization. An innovative protein expression system has been developed by integrating technologies that appear to overcome the major limitations for the manufacturing of mg quantities of recombinant protein, including IMPs, in mammalian cells. Stable, single cell (clonal) lines can be selected to enable controlled over-expression of protein, thereby allowing the selection of an expression system that optimizes specific activity and minimizes aggregation and/or the production of non- functional protein. A novel diagnostic technology provides the ability to rapidly determine optimal solution conditions for protein stability/solubility, reducing unwanted nonspecific aggregation. This same diagnostic can be used to map out solution conditions more likely to result in crystal formation. Specifically, we propose to: 1. Demonstrate the ability to produce milligram quantities of biologically functional membrane proteins from different membrane protein classes in mammalian cells. 2. Demonstrate the utility of a novel diagnostic technology, self-interaction chromatography (SIC), to determine second virial coefficients for different protein solvent conditions as a means to optimize co-solvent combinations and improve protein solubility and stability. 3. Demonstrate the use of SIC as an efficient, knowledge-based approach for the production of diffraction-quality crystals of membrane proteins. The combination of these technologies is expected to improve success rates for determining structures of integral membrane proteins. This project directly addresses several of the current impediments to membrane protein structural biology (outlined on pages 3-6 of this RFA) including protein production, stability, solubility and crystallization. The project involves collaborations with more than fifteen NIH-funded investigators studying different biologically and medically relevant membrane proteins. The project will not only support structural studies performed by the PI, but also provide protein and crystals to other individual crystallographers, NMR spectroscopists and one membrane protein "Specialized Center". Relevance: This work will directly support future efforts to develop new therapeutics as well as enhancing our knowledge of membrane biology. Membrane proteins account for 70% of the drugable market, addressing a wide range of therapeutic categories including pain, asthma, inflammation, obesity, cancer, cardiovascular, metabolic, gastrointestinal and central nervous system diseases cystic fibrosis and more.

描述（由申请人提供）：项目摘要：该项目结合了真核膜蛋白表达，溶解度，稳定和结晶的创新技术。通过整合似乎克服了在哺乳动物细胞中整合Mg重组蛋白（包括IMPS）的重组量的主要局限性的技术，已经开发了一种创新的蛋白质表达系统。可以选择稳定的单细胞（克隆）线以实现对蛋白质的控制过表达，从而允许选择一种表达系统，以优化特定活性并最小化聚集和/或生产非功能性蛋白质。一种新型的诊断技术提供了快速确定蛋白质稳定性/溶解度的最佳解决方案条件的能力，从而减少了不需要的非特异性聚集。相同的诊断可用于绘制溶液条件更有可能导致晶体形成。具体而言，我们建议：1。证明了从哺乳动物细胞中不同膜蛋白类别产生毫克量的生物功能性膜蛋白的能力。 2。证明了一种新型诊断技术，自我交联色谱（SIC）的实用性，以确定不同蛋白质溶剂条件的第二个病毒系数，以此作为优化共溶性组合并改善蛋白质溶解度和稳定性的手段。 3。证明SIC用作一种有效的，基于知识的方法来生产膜蛋白的衍射质量晶体。这些技术的组合有望提高确定整体膜蛋白结构的成功率。该项目直接解决了当前对膜蛋白结构生物学的障碍（在本RFA的第3-6页上概述），包括蛋白质产生，稳定性，溶解度和结晶。该项目涉及与15名NIH资助的研究人员合作，研究了不同的生物学和医学相关的膜蛋白。该项目不仅将支持PI进行的结构研究，还将为其他单独的晶体学家，NMR光谱学家和一个膜蛋白“专业中心”提供蛋白质和晶体。相关性：这项工作将直接支持未来开发新治疗剂的努力，并增强我们对膜生物学的了解。膜蛋白占可药品市场的70％，解决了广泛的治疗类别，包括疼痛，哮喘，炎症，肥胖，癌症，心血管，代谢，胃肠道和中枢神经系统疾病囊性纤维化等。