Fig. 4
Schematic representation of the screening system RVY102. a Cortisone added to the growth medium enters RVY102 cells and is reduced to cortisol by the enzyme 11β-HSD1. The active complex formed by binding of cortisol to GR triggers expression of the TEV protease which binds to its cleavage site and cuts the reporter fluorescent protein that then undergoes degradation. Presence of an 11β-HSD1 inhibitor will stabilize the reporter protein. b Graphic representation of the reporter fusion protein expressed in strain RVY102. The fusion protein consists of the red tandem dimer (td) Tomato, the TEV recognition site, an N-degron destabilizing sequence, the SF3b155381–424 protein domain and the yeast enhanced green fluorescent protein (yEGFP). Once expressed, the TEV protease cuts at its cleavage site on the reporter protein, leading to exposure of the N-degron sequence and degradation of yEGFP. The TEV recognition sequence and the destabilizing amino acid (phenylalanine) are shown