This proposal addresses an RFA # RFA-RM-04-009 initiated by NIH for development of a Center for innovation in membrane protein production. We focus exclusively on eukaryotic membrane proteins since they constitute approximately 30% of all eukaryotic proteins and are targets for over 40% of all drugs in use today, yet there is little understanding of the mechanisms, atomic interactions of these, or even which protein is the target for today's drugs in many cases. Subprojects, each directed by one, or two expert Co-Principle Investigators address eight novel expression systems that will be developed and applied first to strategically selected eukaryotic membrane proteins. These systems range from applying new strategies to prokaryotic expression in E. coil, utilizing L. lactis, utilizing strains of Yeast with expanded endoplasmic reticulum, taking advantage of new technology for suspension culture [and thus higher density] of HEK cells, to the development of a completely novel eukaryotic system, Chlorella. The center will provide continuous assessment of each expression method in its application to each membrane protein, or membrane protein class. Target proteins are used first to establish and calibrate the expression methods. The goal is that the center will ultimately provide access to NIH funded investigators who may arrange to express certain membrane proteins through the center, or who may obtain protocols for application of center methods for their own applications to expression. As expression schemes are evolved and pruned they will each be applied to each membrane protein that enters the center, to select the most efficacious scheme. That method will then be applied to 'industrialized' (meaning standardized, and optimized for routine) expression of that membrane protein. Subprojects also address novel schemes for purification and characterization that have been established as highly successful in initial applications, enhancement of stability of membrane proteins, and generation of, and affinity maturation of antibodies as they may be required for certain applications including some aspects of purification, co-crystallizations, and characterizations. A plan for in vitro production of membrane proteins is to be phased in as the center becomes established. A plan for dissemination of the information gained, up to date results with each novel expression method, purification status with each membrane protein via web access is presented.
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