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

研究成果: Contribution to journalArticle査読

34 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)271-276
ページ数6
ジャーナルBiochemical and Biophysical Research Communications
380
2
DOI
出版ステータス出版済み - 3 6 2009

All Science Journal Classification (ASJC) codes

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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