Production & Crystallization of Membrane Protein for 3D Structure

描述(申请人提供)：本提案解决了真核蛋白表达、增溶、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来实现这一点。这种方法结合了一种新型的高通量自作用层析系统(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维里系数。人工神经网络分析实验得出的第二维里系数数据，并执行对改善蛋白质溶解性和均质性的新溶液条件的电子预测。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知情调整，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重大进展，使其朝着以成本效益、基于知识的方法表达、纯化、稳定和结晶膜蛋白的方向发展。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，这些疾病包括癌症(结肠癌、乳腺癌和前列腺癌)、糖尿病、囊性纤维化、生长异常、免疫系统紊乱、高血压、败血症和流感。对于每个IMP，我们已经与生物化学家/生物学家建立了合作关系，他们对每个目标蛋白质及其蛋白质相互作用组有着长期的兴趣和经验。 与公共健康相关：这项提案旨在开发生产、纯化、稳定和结晶完整膜蛋白的新方案。这将使用几种创新的方法和技术来实现。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、免疫系统紊乱、高血压、败血症和流感。

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.