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, Proteolytic Cleavage of the SARS-CoV-2
Transparent Methods Peptides: Fluorogenic peptides derived from SARS-CoV and SARS-CoV-2 spike (S) S1/S2 sites composed of the sequences HTVSLLRSTSQ and TNSPRRARSVA sequences, respectively, and harboring the (7-methoxycoumarin-4-yl)acetyl/2,4-dinitrophenyl (MCA/DNP) FRET pair were synthesized by Biomatik, Recombinant furin was purchased from New England Biolabs ,
, Recombinant PC1, matriptase, cathepsin B , and cathepsin L were purchased from R&D Systems, Recombinant L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin was obtained from Sigma-Aldrich
, U/mL); 25 mM MES, 5 mM CaCl2, 1% (w/v) Brij-35, pH 6.0 for PC1 (diluted to 2.2 ng/?L); PBS for trypsin (diluted to 8 nM); 50 mM Tris, 50 mM NaCl, 0.01% (v/v) Tween® 20, pH 9.0 for matriptase (diluted to 2.2 ng/?L); 25 mM MES, pH 5.0 for cathepsin B (diluted to 2.2 ng/?L); 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w/v) Brij-35, pH 6.0 for cathepsin L (diluted to 2.2 ng/?L) and with the peptide diluted to 50 ?M. Reactions were performed at 30 °C in triplicates, and fluorescence emission was measured every minute for 45 min using a SpectraMax fluorometer (Molecular Devices, Fluorogenic peptide assay: For each fluorogenic peptide, a reaction was performed in a 100 ?L volume with buffer composed of 100 mM Hepes, 0.5% Triton X-100, 1 mM CaCl2 and 1 mM 2-mercaptoethanol pH 7.5 for furin