Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.
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.
Yurugi-Kobayashi
Takami
T
Iwata Human Receptor Crystallography Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-Ku, Kyoto 606-8501, Japan.
Asada
Hidetsugu
H
Shiroishi
Mitsunori
M
Shimamura
Tatsuro
T
Funamoto
Saeko
S
Katsuta
Naoko
N
Ito
Keisuke
K
Sugawara
Taishi
T
Tokuda
Natsuko
N
Tsujimoto
Hirokazu
H
Murata
Takeshi
T
Nomura
Norimichi
N
Haga
Kazuko
K
Haga
Tatsuya
T
Iwata
So
S
Kobayashi
Takuya
T
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
C487078:CHRM2 protein, human; D043585:Receptor, Muscarinic M2; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins