Innovative Methods for Membrane Protein Crystallization

项目描述（由申请人提供）：项目概述：本项目结合了真核生物膜蛋白表达、增溶、稳定和结晶的创新技术。通过整合技术开发了一种创新的蛋白表达系统，这些技术似乎克服了在哺乳动物细胞中生产mg量重组蛋白（包括IMP）的主要限制。可以选择稳定的单细胞（克隆）系以实现蛋白质的受控过表达，从而允许选择优化比活性并使聚集和/或非功能性蛋白质的产生最小化的表达系统。一种新的诊断技术提供了快速确定蛋白质稳定性/溶解度的最佳溶液条件的能力，减少了不需要的非特异性聚集。这种相同的诊断可以用来绘制出更可能导致晶体形成的溶液条件。具体而言，我们建议：1.证明在哺乳动物细胞中从不同的膜蛋白类中产生毫克量的生物功能膜蛋白的能力。2.演示一种新的诊断技术，自相互作用色谱法（SIC）的效用，以确定不同蛋白质溶剂条件下的第二维里系数，作为优化共溶剂组合和提高蛋白质溶解度和稳定性的一种手段。3.演示SIC作为一种有效的、基于知识的方法用于生产衍射质量的膜蛋白晶体。这些技术的组合有望提高确定膜蛋白结构的成功率。该项目直接解决了目前膜蛋白结构生物学的几个障碍（在本RFA的第3-6页上概述），包括蛋白质生产，稳定性，溶解性和结晶。该项目涉及与超过15个NIH资助的研究人员合作，研究不同的生物和医学相关的膜蛋白。该项目不仅将支持PI进行的结构研究，还将为其他晶体学家，NMR光谱学家和一个膜蛋白“专业中心”提供蛋白质和晶体。相关性：这项工作将直接支持未来开发新疗法的努力，并提高我们对膜生物学的认识。膜蛋白占药物市场的70%，解决了广泛的治疗类别，包括疼痛，哮喘，炎症，肥胖，癌症，心血管，代谢，胃肠道和中枢神经系统疾病囊性纤维化等。

DESCRIPTION (provided by applicant): Project Summary: This project combines innovative technologies in eukaryotic membrane protein expression, solubilization, stablization and crystallization. An innovative protein expression system has been developed by integrating technologies that appear to overcome the major limitations for the manufacturing of mg quantities of recombinant protein, including IMPs, in mammalian cells. Stable, single cell (clonal) lines can be selected to enable controlled over-expression of protein, thereby allowing the selection of an expression system that optimizes specific activity and minimizes aggregation and/or the production of non- functional protein. A novel diagnostic technology provides the ability to rapidly determine optimal solution conditions for protein stability/solubility, reducing unwanted nonspecific aggregation. This same diagnostic can be used to map out solution conditions more likely to result in crystal formation. Specifically, we propose to: 1. Demonstrate the ability to produce milligram quantities of biologically functional membrane proteins from different membrane protein classes in mammalian cells. 2. Demonstrate the utility of a novel diagnostic technology, self-interaction chromatography (SIC), to determine second virial coefficients for different protein solvent conditions as a means to optimize co-solvent combinations and improve protein solubility and stability. 3. Demonstrate the use of SIC as an efficient, knowledge-based approach for the production of diffraction-quality crystals of membrane proteins. The combination of these technologies is expected to improve success rates for determining structures of integral membrane proteins. This project directly addresses several of the current impediments to membrane protein structural biology (outlined on pages 3-6 of this RFA) including protein production, stability, solubility and crystallization. The project involves collaborations with more than fifteen NIH-funded investigators studying different biologically and medically relevant membrane proteins. The project will not only support structural studies performed by the PI, but also provide protein and crystals to other individual crystallographers, NMR spectroscopists and one membrane protein "Specialized Center". Relevance: This work will directly support future efforts to develop new therapeutics as well as enhancing our knowledge of membrane biology. Membrane proteins account for 70% of the drugable market, addressing a wide range of therapeutic categories including pain, asthma, inflammation, obesity, cancer, cardiovascular, metabolic, gastrointestinal and central nervous system diseases cystic fibrosis and more.