(a) Histogram plots of fluorescent ligand-binding flow cytometry data of expressed NTR1, NK1R, and KOR1 variants. In these flow cytometry experiments, the amount of functional receptors at the surface of intact individual cells is determined. Compared is the functional surface expression level of wild-type GPCRs (left panels), library pools obtained after the two rounds of SaBRE (middle panel) and variants evolved in E. coli (right panels). The total signal (red curves) and the nonspecific signal (green, tinted) are shown. For the wild-type GPCRs (NTR1, NK1R, KOR1) no specific signal is obtained, thus no active receptor is detected at the surface. After two rounds of SaBRE, the selected library pools (NTR1 2.5, NK1R 2.5, KOR1 2.5) show a high specific signal, reflecting a high surface expression of functional GPCRs. Variants previously evolved in E. coli (NTR1-D03, NK1R-E11) show a specific signal as well, albeit at significantly lower levels than obtained for the SaBRE library pools and for a significant fraction of cells, no functional expression is detected at the surface (note the double peak of the total signal). For instance, only 50% of the cells express NTR1-D03 at the surface, while for NK1R-E11 only a minority of cells show active surface expression. (b) Measurement of average total functional GPCRs expressed per cell of NTR1, NK1R, and KOR1 variants by radioligand binding. In contrast to flow cytometry analysis, radioligand binding assays account for the total amount of functional receptors averaged across an entire population of lysed cells, and will thus detect functional GPCRs in intracellular membranes as well. The wild-type GPCRs show very low expression levels and the receptor variants previously evolved in E. coli show a low to moderate average functional production. In contrast, the selected SaBRE library pools show high functional expression levels with on average 100,000–150,000 receptors per cell, representing an increase of up to 50- and 20-fold, compared to the wild-type receptors and the variants previously evolved in E. coli, respectively. Error bars indicate standard deviations from triplicates.