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Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.

DOI: 10.1016/j.bbrc.2009.01.053 DOI Help

Authors: Takami Yurugi-kobayashi (Japan Science and Technology Agency) , Hidetsugu Asada (Japan Science and Technology Agency) , Mitsunori Shiroishi (Japan Science and Technology Agency) , Tatsuro Shimamura (Japan Science and Technology Agency) , Saeko Funamoto (Japan Science and Technology Agency) , Naoko Katsuta (Japan Science and Technology Agency) , Keisuke Ito (RIKEN SPring-8 Center) , Taishi Sugawara (University of Tokyo) , Natsuko Tokuda (Kyoto University) , Hirokazu Tsujimoto (Japan Science and Technology Agency) , Takeshi Murata (Japan Science and Technology Agency) , Norimichi Nomura (Japan Science and Technology Agency) , Kazuko Haga (Imperial College London) , Tatsuya Haga (Imperial College London) , Takuya Kobayashi (Japan Science and Technology Agency) , So Iwata (Japan Science and Technology Agency; Kyoto University; Imperial College London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical And Biophysical Research Communications , VOL 380(2) , PAGES 271-6

State: Published (Approved)
Published: March 2009

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.

Journal Keywords: Transporters; Nucleobase:Cation Symporter 1 Family; Membrane Proteins; Hydantoins

Subject Areas: Biology and Bio-materials

Facility: ESRF

Added On: 19/08/2009 23:07

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