DETERMINANTS OF GPCR EXPRESSION IN E COLI AND YEAST

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite the clear and urgent need for better methods to express and study integral membrane proteins, there have been few if any systematic studies to identify the features that lead to the failures to recover high levels of active protein. Grisshammer and Tate (1995) suggested that advances in overexpression systems of membrane proteins required an understanding of how membrane proteins fold and of which cellular proteins are involved in successful folding in the native environment. Our hypothesis has been that only a systematic examination of the molecular and cellular limitations to membrane protein expression for similar families of membrane proteins will lead to improvements in overexpression. This is the focus of the expression component of this subproject, which has two goals: identify the limitations to membrane protein expression in two major expression systems and re-engineer both protein and the expression systems as needed to improve expression. Even with adequate expression levels, the study of membrane proteins is limited by difficulties in extracting and sometimes refolding the proteins. We will therefore also establish a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure. This will include studies of the properties, particularly phase behavior, of the surfactant systems themselves, as well as of their behavior in the presence of membrane proteins. In addition to guiding refolding strategies, this information will be useful in its own right. Our results will provide increased knowledge of the mechanism of membrane protein insertion, folding, and interactions with detergents and lipids, and will facilitate production of significant quantities of active integral membrane proteins.