Fig. 2: Diagram of FACS-based GPCR directed evolution and screen. | Nature Communications

Fig. 2: Diagram of FACS-based GPCR directed evolution and screen.

From: Bridging non-overlapping reads illuminates high-order epistasis between distal protein sites in a GPCR

Fig. 2

a A library of the human adenosine A2a receptor (A2aR) was generated using degenerate NNS codons to perform saturation mutagenesis at five sites within the ligand-binding pocket. Yeast are transformed with the A2aR library and incubated with fluorescent ligand to couple ligand-binding affinity to cellular fluorescence intensity. b The yeast library is screened using fluorescence-activated cell sorting (FACS) to enrich cells producing A2aR variants with high fluorescence intensities. This process is repeated to further enrich receptors with improved ligand-binding affinities while depleting the population of variants with low affinities.

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