Production & Crystallization of Membrane Protein for 3D Structure

生产

• 批准号: 8520338
• 负责人: Lawrence J Delucas
• 金额: $ 28.81万
• 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 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8520338
• 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，溶解度，稳定化和 结晶。我们将通过整合蛋白质质量的早期评估测量和 数量到现有的强大哺乳动物细胞表达平台中。这种方法整合了小说的使用 高通量自我交流色谱系统（HT-SIC）迅速测量第二个病毒 膜蛋白的系数与专门设计的添加剂组合。人造神经 网络分析实验得出的第二个病毒系数数据，并在计算机预测中执行 改善蛋白质溶解度和均匀性的新溶液条件。 HT-SIC系统还可以启用 对改变蛋白质 - 蛋白质相互作用的溶液条件的知情调整，使得 产生高质量的晶体得到改善。实现该提案的具体目标将提供 研究社区具有重大进步，朝着一种经济有效，基于知识的方法进行 表达，纯化，稳定和结晶膜蛋白。 靶蛋白包括两个离子通道蛋白（上皮钠通道，ENAC和囊性） 纤维化跨膜调节蛋白，CFTR），两个GPCR（趋化因子受体-1，CCR1和鞘氨酸 - 1磷酸受体，S1P）和生长激素受体，GHR。这些蛋白质的结构将 为我们对它们的生物学作用机理和在几个 重要疾病包括癌症（结肠，乳腺和前列腺），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。对于每个小鬼，我们都建立了合作 与生物化学家/生物学家一起对每种目标蛋白质的工作和经验长期以来的经验以及 它的蛋白质相互作用。

项目成果

Protein solubilization: a novel approach.

蛋白质溶解：一种新方法。

• DOI: 10.1016/j.jchromb.2014.09.003
• 发表时间: 2014
• 期刊: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
• 作者: Johnson,DavidH;Wilson,WWilliam;DeLucas,LawrenceJ
• 通讯作者: DeLucas,LawrenceJ