Proteinase k

Secreted metabolites in Trichoderma CFs were removed via dialysis (Spectra/Por^® 3 dialysis tube with the molecular weight cut off value of 3.5 kD, Spectrum Laboratories, Rancho Dominguez, CA). After rinsing the dialysis tube with distilled water, it was autoclaved in MilliQ water at 121°C for 15 minutes. Each dialysis tube containing 70 mL CF was placed in 1 L beaker containing 930 mL PDB+LB (1:1) medium. After stirring at 4°C for 5–6 hours, dialysis was repeated twice to obtain secreted metabolite-free CF (labeled as–Met). The growth of 20 bacterial strains, including 19 noted in Table 1 and E. coli, was evaluated by inoculating 10 μL bacterial culture into 2 mL fresh PDB+LB (control) and–Met. After 1–2 days of culturing, the degree of growth inhibition was measured as described above. We used Proteinase K to confirm that some proteins in–Met of T. harzianum exhibit antibacterial activity. Proteinase K solutions in three concentrations (100, 300, and 500 μg/mL) were prepared by mixing 200, 600, and 1000 μg of Proteinase K (Promega, Madison, WI) with 2 mL CF,–Met, and control (fresh PDB+LB). After incubating at 37°C for 2 hours, LR1 and LR3 were cultured in Proteinase K-treated media by shaking (200 rpm) at 25°C for one day. OD₆₀₀ was measured to calculate the degree of growth inhibition. Each treatment consisted of three biological replicates and was repeated three times.

To determine the location (membrane or inside of exosomes) of exosomal THBS2 protein, Proteinase K assay was performed, as described previously 34 (link). 20 mL plasma of 6 LUAD patients were used to purify exosomes (500 μL; concentration: 1062 ng/μL), and were incubated in (1) PBS (control), (2) 1.2 μg/mL Proteinase K (Promega, Catalog Number: V3021; in PBS) alone, (3) 0.5% Triton X-100, or (4) combined Proteinase K and Triton X-100. The samples were then subjected to immunoblot to test TSG-101 (exosomal intra-membrane protein; as positive control), CD9 (exosomal trans-membrane protein; as positive control), THBS2.