Expression System for Isolation of Membrane Associated Protein

描述(由申请人提供)：整体膜蛋白的特性最大的限制是不能分离出足够数量的这种蛋白。真核膜蛋白的分离尤其成问题。我们建议通过开发一系列工具来解决这个问题，以便可靠地表达、检测和纯化完整的膜蛋白。在第一阶段，将构建专门的大肠杆菌宿主菌株和表达载体，以便能够有效地将异源蛋白靶向到大肠杆菌的内膜。我们最近的研究工作已经确定了有希望的N末端伙伴多肽，用于靶向和膜积累模型大肠杆菌蛋白。在第一阶段，我们计划评估这些蛋白融合策略，以实现真核膜蛋白的过量生产和功能膜组装。宿主菌株将携带CPX报告等位基因(见定义)，因此可以监测大肠杆菌的CPX包膜应激反应，以便优化目的蛋白的表达和膜组装。我们还建议证明一种高通量的全细胞ELISA法用于评估感兴趣的融合蛋白的膜积聚。 第二阶段的研究将集中于验证过量生产和分离真核膜受体的表达系统。表达试验将包括对宿主CPX压力报告系统的优势进行彻底的调查。此外，还将使用全细胞ELISA法来评估异源膜蛋白的表达和适当的靶向。膜组件的高通量ELISA分析将与通过常规细胞分离和免疫印迹程序获得的结果相关联。将通过测量全长膜受体的产量来评估菌株的改进(即删除细胞膜蛋白水解酶)。第二阶段还将包括开发改进的蛋白质分离方法。

DESCRIPTION (provided by applicant): The characterization of integral membrane proteins is limited most by the inability to isolate such proteins in sufficient quantities. The isolation of eukaryotic membrane proteins is especially problematic. We propose to address this issue by developing a portfolio of tools for the reliable expression, detection, and purification of integral membrane proteins. In phase I, specialized E. coli host strains and expression vectors will be constructed to enable efficient targeting of heterologous proteins to the inner membrane of E. coli. Our recent research efforts have identified promising N-terminal partner peptides for targeting and membrane accumulation of model E. coli proteins. In phase I, we plan to evaluate these protein fusion strategies for the over-production and functional membrane assembly of a eukaryotic membrane protein. Host strains will carry a Cpx reporter allele (see definitions) so that the Cpx envelope stress response of E. coli may be monitored in order to optimize expression and membrane assembly of the protein of interest. We also propose to demonstrate proof-of- principle for a high-throughput whole-cell ELISA method for assessing membrane accumulation of fusion proteins of interest. Phase II research will focus on validating the expression system for the over-production and isolation of eukaryotic membrane receptors. The expression trials will include a thorough investigation of the advantages of the host Cpx stress reporter system. In addition, the whole- cell ELISA method will be employed to assess expression and proper targeting of heterologous membrane proteins. High-throughput ELISA analysis of membrane assembly will be correlated to results obtained via a conventional cell fractionation and immunoblot procedure. Strain improvements (i.e. deletion of cell envelope proteases) will be evaluated by measuring yield of full-length membrane receptor. Phase II will also include development of improved protein isolation methods.