Expression System for Isolation of Membrane Associated Protein

描述（由申请人提供）：膜整合蛋白的表征最受限制的是无法分离足够量的此类蛋白。真核生物膜蛋白的分离尤其成问题。我们建议通过开发一系列用于可靠表达、检测和纯化完整膜蛋白的工具来解决这个问题。在第一阶段，E.大肠杆菌宿主菌株和表达载体将被构建以使得能够有效地将异源蛋白质靶向至大肠杆菌的内膜。杆菌我们最近的研究工作已经确定了有前途的N-末端伴侣肽的靶向和膜积累的模型E。coli蛋白。在第一阶段，我们计划评估这些蛋白融合策略的过度生产和功能膜组装的真核细胞膜蛋白。宿主菌株将携带Cpx报告等位基因（参见定义），因此Cpx包被E.为了优化目的蛋白的表达和膜组装，可以监测大肠杆菌。我们还提出了一个高通量的全细胞ELISA方法，用于评估膜积累的融合蛋白的目的，以证明的原则。 第二阶段的研究将集中在验证真核细胞膜受体的过度生产和分离的表达系统。表达试验将包括对宿主Cpx应激报告系统优势的彻底调查。此外，将采用全细胞ELISA方法来评估异源膜蛋白的表达和适当靶向。膜组件的高通量ELISA分析将与通过常规细胞分级分离和免疫印迹程序获得的结果相关。将通过测量全长膜受体的产率来评价菌株改良（即细胞包膜蛋白酶的缺失）。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.