%0 Journal Article
%T Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae
%A Mitsunori Shiroishi
%A Hirokazu Tsujimoto
%A Hisayoshi Makyio
%A Hidetsugu Asada
%A Takami Yurugi-Kobayashi
%A Tatsuro Shimamura
%A Takeshi Murata
%A Norimichi Nomura
%A Tatsuya Haga
%A So Iwata
%A Takuya Kobayashi
%J Microbial Cell Factories
%D 2012
%I BioMed Central
%R 10.1186/1475-2859-11-78
%X We 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¨C7£¿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.We 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.
%K G-protein coupled receptor
%K Membrane protein
%K High expression
%K Screening
%K Receptor variants
%K Structural study
%K Saccharomyces cerevisiae
%U http://www.microbialcellfactories.com/content/11/1/78/abstract