Production & Crystallization of Membrane Protein for 3D Structure

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健壮的哺乳动物细胞表达平台中来实现这一目标。该方法集成了一种新型高通量自相互作用色谱系统（HT-SIC）的使用，该系统可以快速测量混合了特殊设计的添加剂的膜蛋白的第二病毒系数。人工神经网络分析了实验得出的第二维里系数数据，并在计算机上预测了改善蛋白质溶解度和均匀性的新溶液条件。HT-SIC系统还可以对改变蛋白质-蛋白质相互作用的溶液条件进行知情调整，从而提高生产高质量晶体的可能性。实现本提案的具体目标将为研究界提供一个具有成本效益，基于知识的方法来表达，纯化，稳定和结晶膜蛋白的重大进展。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GPCRs（趋化因子受体-1，CCR1和鞘氨醇-1磷酸受体，S1P）和生长激素受体，GHR。这些蛋白质的结构将有助于我们理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、免疫系统紊乱、高血压、败血症和流感。对于每个IMP，我们都与生物化学家/生物学家建立了合作关系，他们对每个靶蛋白及其蛋白质相互作用组具有长期的兴趣和经验。

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.