Innovative Methods for Membrane Protein Crystallization

项目概述：本项目结合了真核膜蛋白表达、溶解、稳定和结晶等方面的创新技术。通过整合技术，一种创新的蛋白质表达系统已经开发出来，这些技术似乎克服了在哺乳动物细胞中制造mg数量的重组蛋白（包括imp）的主要限制。可以选择稳定的单细胞（克隆）系来控制蛋白质的过表达，从而允许选择优化特定活性和最小化聚集和/或非功能性蛋白质产生的表达系统。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，减少了不必要的非特异性聚集。同样的诊断也可以用来确定更可能导致晶体形成的溶液条件。具体而言，我们建议：1。演示从哺乳动物细胞的不同膜蛋白类别中产生毫克级生物功能膜蛋白的能力。2. 展示了一种新的诊断技术，自相互作用色谱（SIC）的实用性，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手段。3. 演示使用SIC作为一种高效的，基于知识的方法来生产衍射质量的膜蛋白晶体。这些技术的结合有望提高确定整体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（概述在本RFA的第3-6页），包括蛋白质生产，稳定性，溶解度和结晶。该项目涉及与超过15名美国国立卫生研究院资助的研究人员合作，研究不同的生物学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，还将为其他个体晶体学家、核磁共振波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，并增强我们对膜生物学的了解。膜蛋白占可用药市场的70%，用于广泛的治疗类别，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.