<i>GFP</i> transcription assays to test the selection of affinity-enhanced proteins.

<p>(A–C) Flow cytometry analyses. Fluorescence intensities of the engineered strains expressing cytosolic Z_WT (A), Z_K35A (B) and Z_I31A (C) as the parental ‘Y₂’ proteins. White bars indicate control yeast strains without the expression of ‘Y₁’ (transformed with pGK413 mock vector). (D–F) Fluorescence microscope observations. Fluorescence micrographs of the engineered strains expressing cytosolic Z_WT (D), Z_K35A (E) and Z_I31A (F) as the parental ‘Y₂’ proteins. Five Z variants (ZZ, Z_WT, Z_K35A, Z_I31A and Z₉₅₅) were expressed as the membrane-anchored ‘Y₁’ candidate proteins in each strain. To investigate transmission of the signal, 5 µM of α-factor was used for each strain. Standard errors of three independent experiments are shown.