| Assay Method Information | |
| | high-throughput activity assay |
| Description: | Recombinant SARS-CoV-2 Mpro with native N and C termini was expressed in Escherichia coli, and subsequently purified (Extended Data Fig. 1a, b). The molecular mass of SARS-CoV-2 Mpro as determined by mass spectroscopy is 33797.0 Da, consistent with its theoretical molecular mass of 33796.8 Da. To characterize the enzymatic activity of SARS-CoV-2 Mpro and to carry out high-throughput screening of inhibitors, we developed a fluorescence resonance energy transfer assay. To do this, we designed and synthesized the fluorescently labelled substrate Mca AVLQ↓SGFRK(Dnp)K, derived from the N-terminal autocleavage sequence of the viral protease, for time-dependent kinetic analysis (Extended Data Fig. 1e). The catalytic efficiency (kcat/Km) for SARS-CoV-2 Mpro was measured to be 28,500 M−1 s−1, which is slightly higher than that for SARS-CoV Mpro (kcat/Km = 26,500 M−1 s−1)10 and more than 30-fold higher than that of human rhinovirus 3C protease (kcat/Km = 920 M−1 s−1). |
| Affinity data for this assay | |
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