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Background  Various protein expression systems, such as  Escherichia coli  ( E. coli ),  Saccharomyces cerevisiae  ( S. cerevisiae ),  Pichia pastoris  ( P. pastoris ), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β 2  adrenergic receptor, adenosine A 2a  receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study,  P. pastoris  and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.    Results  The ideal conditions for the expression of CHRM2 in  P. pastoris  were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [ 3 H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by  P. pastoris  was lower than that of Sf9 insect cells, CHRM2 yield in  P. pastoris  was 2-fold higher than in Sf9 insect cells because  P. pastoris  was cultured at high cell density. The dissociation constant (Kd) for QNB in  P. pastoris  was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between  P. pastoris  and Sf9 insect cells.    Conclusion  Compared to insect cells,  P. pastoris  is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast,  P. pastoris , and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in  P. pastoris  can be applied to the structural and biochemical studies of GPCRs.

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis