TY - JOUR
T1 - Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris
AU - Yurugi-Kobayashi, Takami
AU - Asada, Hidetsugu
AU - Shiroishi, Mitsunori
AU - Shimamura, Tatsuro
AU - Funamoto, Saeko
AU - Katsuta, Naoko
AU - Ito, Keisuke
AU - Sugawara, Taishi
AU - Tokuda, Natsuko
AU - Tsujimoto, Hirokazu
AU - Murata, Takeshi
AU - Nomura, Norimichi
AU - Haga, Kazuko
AU - Haga, Tatsuya
AU - Iwata, So
AU - Kobayashi, Takuya
N1 - Funding Information:
This research was supported in part by a Research Grant from the ERATO Iwata Human Receptor Crystallography Project from the Japan Science and Technology Agency (JST) to S.I.; a Research Fellowship from the Uehara Memorial Foundation to T.K.; a Long-term Research Grants from the Toyobo Biotechnology Foundation to T.K.; a Research Grant Abroad from the Japan Heart Foundation/Bayer Yakuhin to T.Y.-K.
PY - 2009/3/6
Y1 - 2009/3/6
N2 - 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.
AB - 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.
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U2 - 10.1016/j.bbrc.2009.01.053
DO - 10.1016/j.bbrc.2009.01.053
M3 - Article
C2 - 19167344
AN - SCOPUS:60349124153
SN - 0006-291X
VL - 380
SP - 271
EP - 276
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -