Innovative Methods for Membrane Protein Crystallization

描述(申请人提供)：项目概述：该项目结合了真核膜蛋白表达、溶解、稳定和结晶方面的创新技术。通过整合似乎克服了在哺乳动物细胞中制造包括IMPS在内的大量重组蛋白的主要限制的技术，开发了一种创新的蛋白质表达系统。可以选择稳定的单细胞(克隆)系以实现受控的蛋白质过度表达，从而允许选择优化特定活性并使非功能蛋白质的聚集和/或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/溶解性的最佳溶液条件的能力，减少了不必要的非特异性聚集。同样的诊断可以用来绘制出更有可能导致晶体形成的溶液条件。具体地说，我们建议：1.证明在哺乳动物细胞中从不同的膜蛋白类别中产生毫克量生物功能膜蛋白的能力。2.证明了一种新的诊断技术--自作用层析(SIC)用于测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、提高蛋白质的溶解性和稳定性的手段。3.展示了使用碳化硅作为一种有效的、基于知识的方法来生产膜蛋白的衍射级晶体。这些技术的结合有望提高确定完整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页中概述)，包括蛋白质生产、稳定性、溶解性和结晶。该项目涉及与NIH资助的15多名研究人员合作，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，还将向其他个人结晶学家、核磁共振光谱仪和一个膜蛋白质“专门中心”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，以及增强我们对膜生物学的知识。膜蛋白占可用药市场的70%，涉及范围广泛的治疗类别，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.