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

Mitsunori Shiroishi, Hirokazu Tsujimoto, Hisayoshi Makyio, Hidetsugu Asada, Takami Yurugi-Kobayashi, Tatsuro Shimamura, Takeshi Murata, Norimichi Nomura, Tatsuya Haga, So Iwata, Takuya Kobayashi

研究成果: ジャーナルへの寄稿記事

33 引用 (Scopus)

抄録

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.

元の言語英語
記事番号78
ジャーナルMicrobial Cell Factories
11
DOI
出版物ステータス出版済み - 6 13 2012

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Crystallography
G-Protein-Coupled Receptors
Yeast
Saccharomyces cerevisiae
Proteins
Screening
Pichia
Crystallization
Purification

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

これを引用

Shiroishi, M., Tsujimoto, H., Makyio, H., Asada, H., Yurugi-Kobayashi, T., Shimamura, T., ... Kobayashi, T. (2012). Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. Microbial Cell Factories, 11, [78]. https://doi.org/10.1186/1475-2859-11-78

Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. / Shiroishi, Mitsunori; Tsujimoto, Hirokazu; Makyio, Hisayoshi; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Murata, Takeshi; Nomura, Norimichi; Haga, Tatsuya; Iwata, So; Kobayashi, Takuya.

:: Microbial Cell Factories, 巻 11, 78, 13.06.2012.

研究成果: ジャーナルへの寄稿記事

Shiroishi, M, Tsujimoto, H, Makyio, H, Asada, H, Yurugi-Kobayashi, T, Shimamura, T, Murata, T, Nomura, N, Haga, T, Iwata, S & Kobayashi, T 2012, 'Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae', Microbial Cell Factories, 巻. 11, 78. https://doi.org/10.1186/1475-2859-11-78
Shiroishi, Mitsunori ; Tsujimoto, Hirokazu ; Makyio, Hisayoshi ; Asada, Hidetsugu ; Yurugi-Kobayashi, Takami ; Shimamura, Tatsuro ; Murata, Takeshi ; Nomura, Norimichi ; Haga, Tatsuya ; Iwata, So ; Kobayashi, Takuya. / Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. :: Microbial Cell Factories. 2012 ; 巻 11.
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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.",
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AU - Yurugi-Kobayashi, Takami

AU - Shimamura, Tatsuro

AU - Murata, Takeshi

AU - Nomura, Norimichi

AU - Haga, Tatsuya

AU - Iwata, So

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