Figure 1

Overview of the construction and evaluation platform of GPCR variants in S. cerevisiae. (A) Primer design of a variant. As an example, the hHRH1 variant with truncation of the N-terminal region, a mutation at the 3.41 position in TM3 and T4L fusion to the i3-loop is shown. The four PCR fragments are generated using the indicated primer pairs from full-length GPCR and T4L ( Additional file1: Table S1). The same colored overlapping regions were necessary for homologous recombination in S. cerevisiae. (B) Illustration of a cycle from construction design to evaluation for the GPCR variants. (C) Flow and time-scale of the construction and evaluation of GPCR variants in three hosts. Schemes for small-scale culturing are shown in yellow and orange, respectively. Evaluations by ligand-binding assays and FSEC are shown in cyan and green, respectively. Vec.: preparation of expression vector, Bac.: preparation of Bacmid, Ura-: selection on a Ura- plate, MD: selection on a minimum dextrose (MD) plate, Gen: selection on a geneticine plate, P1 and P2: P1 and P2 virus preparation, respectively.