Fig. 3.

Engineering and evaluation of bacteria-displayed pro-Affibody molecules. a Representative results from flow-cytometric analysis of efficiency of masking and proteolytic activation of different linkers connecting the two domains of the pro-Affibody. The protease substrate is flanked by one (green), two (red) or three (blue) G₄S repeats. Light colors represent the non-activated and dark colors the activated pro-Affibody. b Representative results from flow-cytometric analysis of two different substrate peptides, GPQAIAGQ (green) and GPQGIAGQ (gray). Pro-Affibody molecules containing the substrates were tested for their ability to be activated by MMP-1. c Representative results from flow-cytometric analysis of negative control pro-Affibody (Z_HER2 fused to Z_HER3). The HER2-binding Affibody molecule was fused to a non-binding Affibody molecule (Z_HER3) as the “masking” domain, which did not mask before proteolysis (purple) and was not affected by proteolysis (black). Shown in green is the non-activated pro-Affibody with the masking domain (anti-Z_HER2). d Binding to HER2 of activated pro-Affibody (green) and non-activated pro-Affibody (red). HER2 concentration was titrated from 0.1 to 1,000 nM. Dots represent mean values (±SD) of MFI measured by flow cytometry from two independent experiments. Solid line represents fitted data by non-linear regression to a 1:1 binding model. All experiments were performed at least in duplicates on different days using freshly prepared samples and reagents