Figure 3: Selection of cathepsin E-activity-enhancing peptides at neutral pH. (a) Selection products from the primary library and the secondary library. For both, the top 3 peptides are shown. For the activity assay, the peptides used here were synthesized by an in vitro translation method [18]. Here, the ratio of CatE to an activity-enhancing peptide was 1/1 (= 20 nM/20 nM (see Materials and Methods for detail). Each error bar represent average and standard deviation values of three independent experiments. (b) Biacore sensorgrams of peptides, P3 (primary library selection product) and S3 (secondary library one) against cathepsin E (3.36 mg/mL) immobilized onto sensor chip CM5. To determine dissociation constants, four different concentrations (25, 12.5, 6.3, and 3.2 μM for P3 and 50, 25, 12.5, and 6.3 μM for S3) of the peptides were subjected to the interaction. * 𝑃 < . 0 8 , ** 𝑃 < . 0 5 , and *** 𝑃 < . 0 0 0 3 compared with the control group CatE and substrate only (without peptide activator), by Student’s 𝑡 -test.