Tuning expression of GPCRs for the secretory pathway in the baculovirus-insect cell expression system.

Abstract

The overexpression of G-protein-coupled receptors (GPCRs) remains one of the biggest hurdles for structural studies of these proteins. To date, the most usually applied system for this task is the insect cell/baculovirus expression system. A drawback of this system, however, is the accumulation of protein that is resistant to solubilization with the commonly used mild detergent DoDecylMaltoside (DDM). In addition, poor surface expression is often observed. In this study, it is shown how an earlier AcMNPV 39K promoter, can express receptors that are found primarily on the cell membrane, as revealed by confocal microscopy, and the protein can be solubilized to a higher degree by DDM in a less aggregation-prone form, as monitored by fluorescence size-exclusion chromatography. In addition, a strong effect on the yield was observed when the AcMNPV gp67 signal sequence was used. The documentation of the 39K promoter as an improvement over the frequently used polyhedrin promoter, along with the effect of the gp67 signal sequence are important steps toward ultimately improving the expression in terms of total functional yield, while also shedding light on the nature of the process of overproduction of membrane proteins, in particular, GPCRs.