Production & Crystallization of Membrane Protein for 3D Structure

生产

• 批准号: 8028213
• 负责人: Lawrence J Delucas
• 金额: $ 30万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 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/8028213
• 关键词: Address; Biological; Biological Neural Networks; Breast; CCR1 gene; Charge; Chemicals; Chromatography; Collaborations; Colon Carcinoma; Communication; Communities; Comprehensive Cancer Center; Computer Simulation; Core Facility; Crystallization; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Diabetes Mellitus; Disease; Effectiveness; Epithelial; Excipients; Fee-for-Service Plans; Growth; Growth Hormone Receptor; Human Resources; Hypertension; Immune System Diseases; Information Systems; Integral Membrane Protein; Ion Channel Protein; Laboratories; Mammalian Cell; Manuals; Measurement; Measures; Membrane; Membrane Proteins; Outcome; Outcomes Research; Pathway Analysis; Probability; Production; Prostate; Proteins; Protocols documentation; Publications; Publishing; Research; Research Personnel; Roentgen Rays; Role; Sepsis; Sodium Channel; Solubility; Solutions; Sphingosine-1-Phosphate Receptor; Structure; Supervision; System; Technology; Work; abstracting; base; chemokine receptor; cost; cost effective; design; epithelial Na+ channel; experience; flu; human disease; improved; innovation; interest; knowledge base; meetings; milligram; new technology; novel; prevent; protein complex; protein expression; protein protein interaction; protein purification; protein structure; success; three dimensional structure; web site

DESCRIPTION (provided by applicant): This proposal addresses challenges in eukaryotic protein expression, solubilization, stabilization and crystallization. We will accomplish this by integrating early assessment measurements of protein quality and quantity into an existing robust mammalian cell expression platform. This approach integrates use of a novel high-throughput self-interaction chromatography system (HT-SIC) that rapidly measures second virial coefficients for the membrane protein mixed with a specially designed panel of additives. An artificial neural network analyzes the experimentally derived second virial coefficient data and performs in silico predictions of novel solution conditions that improve protein solubility and homogeneity. The HT-SIC system also enables informed adjustments to solution conditions that alter protein-protein interactions such that the probability of producing high-quality crystals is improved. Achieving the specific aims of this proposal will provide the research community with significant advancements toward a cost effective, knowledge-based approach to express, purify, stabilize and crystallize membrane proteins. Target proteins include two ion channel proteins (epithelial sodium channel, ENaC and cystic fibrosis transmembrane regulator protein, CFTR), two GPCRs (chemokine receptor-1, CCR1 and sphingosine- 1 phosphate receptor, S1P) and growth hormone receptor, GHR. Structures of these proteins would contribute significantly to our understanding of their biological mechanism of action and role in several important diseases including cancer (colon, breast and prostate), diabetes, cystic fibrosis, growth anomalies, immune system disorders, hypertension, sepsis and the flu. For each IMP, we have established collaborations with biochemists/biologists with a long-standing interest in and experience working with each target protein and its protein interactome. PUBLIC HEALTH RELEVANCE: This proposal is directed at the development of novel protocols to produce, purify, stabilize and crystallize integral membrane proteins. This will be accomplished using several innovative approaches and technologies. The proteins targeted are directly associated with human disease including cancer (colon, breast and prostate), diabetes, cystic fibrosis, growth anomalies, immune system disorders, hypertension, sepsis and the flu.

描述（由申请人提供）：该提案解决了真核蛋白表达、溶解、稳定和结晶方面的挑战。我们将通过将蛋白质质量和数量的早期评估测量整合到现有的强大的哺乳动物细胞表达平台中来实现这一目标。这种方法集成了使用一种新的高通量自相互作用色谱系统（HT-SIC），快速测量第二维里系数的膜蛋白与一个专门设计的面板的添加剂混合。人工神经网络分析了实验得出的第二维里系数数据，并在计算机模拟中预测了改善蛋白质溶解度和均匀性的新溶液条件。HT-SIC系统还能够对改变蛋白质-蛋白质相互作用的溶液条件进行明智的调整，从而提高生产高质量晶体的可能性。实现这一建议的具体目标将为研究界提供一个具有成本效益的，以知识为基础的方法来表达，纯化，稳定和结晶膜蛋白的显着进步。 靶蛋白包括两种离子通道蛋白（上皮钠通道，ENaC和囊性纤维化跨膜调节蛋白，CFTR），两种GPCR（趋化因子受体-1，CCR 1和鞘氨醇-1磷酸受体，S1 P）和生长激素受体，GHR。这些蛋白质的结构将大大有助于我们了解它们在几种重要疾病中的生物学作用机制和作用，包括癌症（结肠癌、乳腺癌和前列腺癌）、糖尿病、囊性纤维化、生长异常、免疫系统疾病、高血压、败血症和流感。对于每种IMP，我们都与生物化学家/生物学家建立了合作关系，这些生物化学家/生物学家对每种靶蛋白及其蛋白质相互作用组具有长期的兴趣和经验。 公共卫生关系：该建议是针对开发新的协议，以生产，纯化，稳定和结晶的完整的膜蛋白。这将通过采用若干创新办法和技术来实现。靶向的蛋白质与人类疾病直接相关，包括癌症（结肠癌、乳腺癌和前列腺癌）、糖尿病、囊性纤维化、生长异常、免疫系统疾病、高血压、败血症和流感。