title: Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.
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 B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.
authors.affiliation: Iwata Human Receptor Crystallography Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Takami
authors.initials: T
authors.lastname: Yurugi-Kobayashi
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Saeko
authors.initials: S
authors.lastname: Funamoto
authors.forename: Naoko
authors.initials: N
authors.lastname: Katsuta
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Taishi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tokuda
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsujimoto
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
authors.forename: Kazuko
authors.initials: K
authors.lastname: Haga
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D006031:Glycosylation; D006801:Humans; D010843:Pichia; D014176:Protein Biosynthesis; D043585:Receptor, Muscarinic M2 / Q000096:biosynthesis* / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: C487078:CHRM2 protein, human; D043585:Receptor, Muscarinic M2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins