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. This is clearly directly related to the paucity of membrane protein structures. A recent review of heterologous expression of an important class of mammalian membrane proteins, the G-protein coupled receptors, tabulates expression levels in E. coli, yeast, insect cell, and mammalian systems. Of 165 reported studies of heterologous GPCR expression, only 2 examples yielded protein at levels greater than 1 mg/5 L, prior to purification attempts. For the purposes of this proposal, a minimal expression goal will be 1 mg/L, where production of 100 mg or more in any expression system reaches an ideal case. Why has overexpression of membrane proteins been so low? A decade ago, expression attempts were fewer, and many believed that an empirical study of expression systems would prove to identify the cause. However, as early as 1995, Grisshammer and Tate suggested that advances in overexpression systems of membrane proteins would only result when we understood how membrane proteins fold and which cellular proteins were involved in successful folding in the native environment. This situation is virtually unchanged in almost a decade. 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 this subproject. The goals of this subproject are two-fold: 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.