Abstract
BackgroundRecent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.ResultsWe developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.ConclusionsWe demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
Highlights
Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification
We demonstrate that the platform using S. cerevisiae is very useful for the rapid construction and evaluation of GPCR variants for structural study
The clone harboring the GPCR variant is selected on an agar plate without uracil (Ura-)
Summary
Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. The high-resolution 3D structures of the target GPCRs provide good initial models for drug design, difficulties in expression and purification have been a major receptor in order to achieve high expression and/or facilitation of crystallization [7,8,9,10,11,12,13]. E. coli has recently been used to screen the thermally stable GPCR variants of turkey β1 adrenergic receptor (tADRB1) [14], human adenosine A2a receptor (hADORA2A) [15, 16], and rat neurotensin receptor 1 (rNTSR1) [17]. Only a limited number of functionally expressed receptors have been successfully generated in E. coli [20]
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