Research Project
7404781
[unreadable] 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. [unreadable] [unreadable] 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. [unreadable] [unreadable] [unreadable]
