Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

Hidetsugu Asada, Tomoko Uemura, Takami Yurugi-Kobayashi, Mitsunori Shiroishi, Tatsuro Shimamura, Hirokazu Tsujimoto, Keisuke Ito, Taishi Sugawara, Takanori Nakane, Norimichi Nomura, Takeshi Murata, Tatsuya Haga, So Iwata, Takuya Kobayashi

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Background: Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.Results: The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.Conclusion: Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

Original languageEnglish
Article number24
JournalMicrobial Cell Factories
Volume10
DOIs
Publication statusPublished - Apr 22 2011

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Pichia
G-Protein-Coupled Receptors
Structural analysis
Quinuclidinyl Benzilate
Insects
Proteins
Yeast
Sf9 Cells
Crystal structure
Dopamine D3 Receptors
Purinergic P1 Receptors
Mammals
Adrenergic Receptors
Escherichia coli
Assays
Buffers
Membrane Proteins
Cells
Yeasts
Costs

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Asada, H., Uemura, T., Yurugi-Kobayashi, T., Shiroishi, M., Shimamura, T., Tsujimoto, H., ... Kobayashi, T. (2011). Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. Microbial Cell Factories, 10, [24]. https://doi.org/10.1186/1475-2859-10-24

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. / Asada, Hidetsugu; Uemura, Tomoko; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Shimamura, Tatsuro; Tsujimoto, Hirokazu; Ito, Keisuke; Sugawara, Taishi; Nakane, Takanori; Nomura, Norimichi; Murata, Takeshi; Haga, Tatsuya; Iwata, So; Kobayashi, Takuya.

In: Microbial Cell Factories, Vol. 10, 24, 22.04.2011.

Research output: Contribution to journalArticle

Asada, H, Uemura, T, Yurugi-Kobayashi, T, Shiroishi, M, Shimamura, T, Tsujimoto, H, Ito, K, Sugawara, T, Nakane, T, Nomura, N, Murata, T, Haga, T, Iwata, S & Kobayashi, T 2011, 'Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis', Microbial Cell Factories, vol. 10, 24. https://doi.org/10.1186/1475-2859-10-24
Asada, Hidetsugu ; Uemura, Tomoko ; Yurugi-Kobayashi, Takami ; Shiroishi, Mitsunori ; Shimamura, Tatsuro ; Tsujimoto, Hirokazu ; Ito, Keisuke ; Sugawara, Taishi ; Nakane, Takanori ; Nomura, Norimichi ; Murata, Takeshi ; Haga, Tatsuya ; Iwata, So ; Kobayashi, Takuya. / Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. In: Microbial Cell Factories. 2011 ; Vol. 10.
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