Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris

Takami Yurugi-Kobayashi, Hidetsugu Asada, Mitsunori Shiroishi, Tatsuro Shimamura, Saeko Funamoto, Naoko Katsuta, Keisuke Ito, Taishi Sugawara, Natsuko Tokuda, Hirokazu Tsujimoto, Takeshi Murata, Norimichi Nomura, Kazuko Haga, Tatsuya Haga, So Iwata, Takuya Kobayashi

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a Bmax value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

Original languageEnglish
Pages (from-to)271-276
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume380
Issue number2
DOIs
Publication statusPublished - Mar 6 2009

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Glycosylation
Pichia
G-Protein-Coupled Receptors
Membrane Proteins
Clone Cells
Muscarinic Receptors
Post Translational Protein Processing
Crystallization
Yeast
Assays
Proteins
Yeasts
Ligands
Temperature

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Yurugi-Kobayashi, T., Asada, H., Shiroishi, M., Shimamura, T., Funamoto, S., Katsuta, N., ... Kobayashi, T. (2009). Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris. Biochemical and Biophysical Research Communications, 380(2), 271-276. https://doi.org/10.1016/j.bbrc.2009.01.053

Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris. / Yurugi-Kobayashi, Takami; Asada, Hidetsugu; Shiroishi, Mitsunori; Shimamura, Tatsuro; Funamoto, Saeko; Katsuta, Naoko; Ito, Keisuke; Sugawara, Taishi; Tokuda, Natsuko; Tsujimoto, Hirokazu; Murata, Takeshi; Nomura, Norimichi; Haga, Kazuko; Haga, Tatsuya; Iwata, So; Kobayashi, Takuya.

In: Biochemical and Biophysical Research Communications, Vol. 380, No. 2, 06.03.2009, p. 271-276.

Research output: Contribution to journalArticle

Yurugi-Kobayashi, T, Asada, H, Shiroishi, M, Shimamura, T, Funamoto, S, Katsuta, N, Ito, K, Sugawara, T, Tokuda, N, Tsujimoto, H, Murata, T, Nomura, N, Haga, K, Haga, T, Iwata, S & Kobayashi, T 2009, 'Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris', Biochemical and Biophysical Research Communications, vol. 380, no. 2, pp. 271-276. https://doi.org/10.1016/j.bbrc.2009.01.053
Yurugi-Kobayashi T, Asada H, Shiroishi M, Shimamura T, Funamoto S, Katsuta N et al. Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris. Biochemical and Biophysical Research Communications. 2009 Mar 6;380(2):271-276. https://doi.org/10.1016/j.bbrc.2009.01.053
Yurugi-Kobayashi, Takami ; Asada, Hidetsugu ; Shiroishi, Mitsunori ; Shimamura, Tatsuro ; Funamoto, Saeko ; Katsuta, Naoko ; Ito, Keisuke ; Sugawara, Taishi ; Tokuda, Natsuko ; Tsujimoto, Hirokazu ; Murata, Takeshi ; Nomura, Norimichi ; Haga, Kazuko ; Haga, Tatsuya ; Iwata, So ; Kobayashi, Takuya. / Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris. In: Biochemical and Biophysical Research Communications. 2009 ; Vol. 380, No. 2. pp. 271-276.
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