Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris

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• 作者：Takami Yurugi-Kobayashi ; Hidetsugu Asada ; Mitsunori Shiroishi ; Tatsuro Shimamura ; Saeko Funamoto ; Naoko Katsuta ; Keisuke Ito ; Taishi Sugawara ; Natsuko Tokuda ; Hirokazu Tsujimoto ; Takeshi Murata ; Norim
• 关键词：Membrane protein ; Pichia pastoris ; Structural biology ; G-protein-coupled receptor (GPCR) ; Non-glycosylation
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2009
• 出版时间：6 March 2009
• 年：2009
• 卷：380
• 期：2
• 页码：271-276
• 全文大小：376 K

文摘

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.