Production & Crystallization of Membrane Protein for 3D Structure

生产

• 批准号: 8309973
• 负责人: Lawrence J Delucas
• 金额: $ 29.7万
• 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/8309973
• 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

Abstract: This proposal addresses challenges in eukaryotic protein expression, solublization, stablization 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.

摘要：该提案解决了真核蛋白表达、溶解、稳定和 结晶。我们将通过整合蛋白质质量的早期评估测量和 数量进入现有的强大的哺乳动物细胞表达平台。这种方法结合了一种新颖的 高通量自相互作用色谱系统 (HT-SIC)，可快速测量第二维里 膜蛋白与专门设计的添加剂组混合的系数。人工神经元 网络分析实验得出的第二维里系数数据并进行计算机预测 提高蛋白质溶解度和均匀性的新颖溶液条件。 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，使得 提高了生产高质量晶体的能力。实现本提案的具体目标将提供 研究界在成本效益、基于知识的方法方面取得了重大进展 表达、纯化、稳定和结晶膜蛋白。 靶蛋白包括两种离子通道蛋白（上皮钠通道、ENaC 和囊性通道蛋白） 纤维化跨膜调节蛋白 (CFTR)、两种 GPCR（趋化因子受体 1、CCR1 和鞘氨醇） 1 磷酸盐受体 (S1P) 和生长激素受体 (GHR)。这些蛋白质的结构 极大地有助于我们了解其作用的生物学机制和在多个方面的作用 重要疾病，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。对于每个 IMP，我们都建立了合作关系 与对每种目标蛋白长期感兴趣并具有相关工作经验的生物化学家/生物学家合作， 它的蛋白质相互作用组。