Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae

Microbial Cell Factories

Research

Tatsuya Haga¹  Takeshi Murata²  Norimichi Nomura²  Hisayoshi Makyio²  Takami Yurugi-Kobayashi³  Hidetsugu Asada³  Hirokazu Tsujimoto³  Tatsuro Shimamura³  So Iwata⁴  Takuya Kobayashi⁵  Mitsunori Shiroishi⁶ 

[1] Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, 171-8588, Toshima-ku, Tokyo, Japan;Iwata Human Receptor Crystallography project, ERATO, JST, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Iwata Human Receptor Crystallography project, ERATO, JST, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Department of Medical Chemistry and Cell Biology, Kyoto University Faculty of Medicine, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Iwata Human Receptor Crystallography project, ERATO, JST, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Department of Medical Chemistry and Cell Biology, Kyoto University Faculty of Medicine, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Division of Molecular Biosciences Membrane, Protein Crystallography Group, Imperial College London, SW7 2AZ, London, UK;Iwata Human Receptor Crystallography project, ERATO, JST, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Department of Medical Chemistry and Cell Biology, Kyoto University Faculty of Medicine, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Japan Science and Technology Agency, Core Research for Evolution Science and Technology (CREST), Kyoto University Faculty of Medicine, 606-8501, Kyoto, Japan;Iwata Human Receptor Crystallography project, ERATO, JST, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, 812-8582, Higashi-ku, Fukuoka, Japan;Department of Medical Chemistry and Cell Biology, Kyoto University Faculty of Medicine, Yoshidakonoe-cho, 606-8501, Sakyo-ku, Kyoto, Japan;

关键词: G-protein coupled receptor;    Membrane protein;    High expression;    Screening;    Receptor variants;    Structural study;    Saccharomyces cerevisiae;   

DOI  :  10.1186/1475-2859-11-78

 received in 2012-02-24, accepted in 2012-05-20,  发布年份 2012

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Shiroishi et al.; licensee BioMed Central Ltd. 2012