Fig. 1: Cell-based screening process identifies CA2-DRD variants with drug-responsive stability.

a Variant libraries were generated through a combination of random and/or saturation mutagenesis and structure-guided engineering. DNA libraries were fused to a GFP or Thy1.2 expression cassette and integrated into Jurkat cells via lentiviral transduction. Iterative fluorescence-activated cell sorting was used to select for library members showing ACZ ligand-dependent expression of GFP or Thy1.2. Once an enriched library with desired characteristics was obtained, single-cell sorting was used to identify CA2-DRD variants with favorable properties (low basal expression, high dynamic range) for further characterization. Representative library data from CA2-Thy1.2 screen is shown. Characterization of single-cell clones from GFP screen shown. b CA2-DRD variants identified by cell-based screening process were fused to GFP and expressed in Jurkat cells by lentiviral transduction. Cells were treated with a range of ACZ concentrations and GFP MFI was measured in transduced cells by flow cytometry. c Dose-response curves were used to calculate regulation parameters (EC₅₀, EC₉₀, fold-change [FC= [GFP MFI]_ACZ/[GFP MFI]_DMSO]). Error bars show standard error of the mean (N = 3).