Figure 1

Schema of GPCR expression in P. pastoris and Sf9 insect cells and human CHRM2 mutant. The CHRM2-expressing plasmid vector was constructed (Vac.). In the P. pastoris expression system, the plasmid was transformed into the P. pastoris strain SMD1163. The transformants were inoculated onto MD plates (MD) and selected by YPD agar plates containing G418 (0.1 or 0.25 mg/mL) (Gen). In the Sf9 insect cell expression system, baculovirus was obtained by homologous recombination in E. coli using the Bac-to-Bac baculovirus expression system (Invitrogen) (Bac.). Baculoviruses were amplified stepwise (P1 and P2). Small- and large-scale cultures are shown in yellow and orange, respectively. Evaluation by a ligand-binding assay is represented in green (A). Four N-linked glycosylation sites in the N-terminus were eliminated by converting asparagine residues (Asn2, 3, 6 and 9) to aspartic acid (Asp). Amino acid residues 234- 381 of the third intracellular loop of the human CHRM2 were deleted (B).