Protease inhibitors set

S. aureus strains and plasmids used in this work are listed in Table 1. AH4654 is one of 75 clinical isolates, isolated from 10 pediatric CF patients and kindly gifted by the Starner Lab, University of Iowa. S. aureus was cultured in tryptic soy broth (TSB) or brain heart infusion (BHI) broth, and E. coli was cultured in lysogeny broth (LB) at 37°C with shaking at 200 rpm. Antibiotics were added to the media at the following concentrations: chloramphenicol (Cam), 10 µg/mL; erythromycin (Erm), 5 µg/mL; and tetracycline (Tet), 1 µg/mL. E. coli strains with plasmids were maintained on media supplemented with ampicillin at 100 µg/mL; kanamycin at 50 µg/mL; or spectinomycin at 50 µg/mL. Porcine trypsin and the Protease Inhibitors Set (Roche) were purchased from Sigma. Recombinant proteases (human trypsin, mouse kallikrein 1, and airway trypsin-like protease) were purchased from G-Biosciences, Sigma-Aldrich, and ThermoFisher Scientific (Biotechne). Stimulated saliva was collected over 10–30 min by chewing on paraffin wax. Particulate material was removed by centrifugation, and this clarified saliva was stored at 4°C for up to 2 days.

The effect of protease inhibitors on the proteolytic activity of the SK1-1 crude enzyme was examined using three individual protease inhibitors from the Protease Inhibitors Set (Sigma). The crude enzymes were treated with leupeptin (at working concentrations of 1.5 and 3 μg/mL), pepstatin (PS; 7.5 and 15 μg/mL), and Pefabloc^® SC (PSC; 0.5 and 1 mg/mL). The solution of each protease inhibitor was prepared in appropriate solvents according to the manufacturer’s instructions. The crude enzyme was mixed with each protease inhibitor and incubated at room temperature (23 °C) for 30 min. Then, the mixture was reacted with azocasein at 37 °C for 2 h. The proteolytic activity of the crude enzyme without protease inhibitor treatment was considered to be the control (100%).