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Open Access Research

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

Mitsunori Shiroishi123*, Hirokazu Tsujimoto13, Hisayoshi Makyio1, Hidetsugu Asada13, Takami Yurugi-Kobayashi13, Tatsuro Shimamura13, Takeshi Murata1, Norimichi Nomura1, Tatsuya Haga4, So Iwata135* and Takuya Kobayashi136*

Author Affiliations

1 Iwata Human Receptor Crystallography project, ERATO, JST, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

2 Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

3 Department of Medical Chemistry and Cell Biology, Kyoto University Faculty of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

4 Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan

5 Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, London, SW7 2AZ, UK

6 Japan Science and Technology Agency, Core Research for Evolution Science and Technology (CREST), Kyoto University Faculty of Medicine, Kyoto, 606-8501, Japan

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Microbial Cell Factories 2012, 11:78  doi:10.1186/1475-2859-11-78

Published: 13 June 2012

Abstract

Background

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. Therefore, the quick screening of functionally expressed stable receptor variants is vital.

Results

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 6–7 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.

Conclusions

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.

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