| Assay Method Information | |
| | Enzyme Activity Assay |
| Description: | Assay details: A SARS-Cov-2 Mpro construct was prepared, which includes a His-tag and PreScission cleavage site (see FIG. 3A). The construct was transformed into E. coli and the enzyme was isolated using Ni2+-affinity chromatography. The C-terminal His tag was cleaved by PreScission and the N-term was autocleaved by the protease itself. The protein was further purified by FPLC and purity was assessed by gel electrophoresis and Coomassie staining. The enzymatic activity of purified SARS-CoV-2 Mpro was tested using a dabcyl-KTSAVLQ↓SGFRKME(Edans)-NH2 substrate (GL Biochem) by monitoring at em 460 nm with ex 360 nm (the assay is schematically shown in FIG. 3B, adapted from Kasperkeiwicz et al., FEBS J, 284 (10), 1518-1539). Initial velocities were determined from the linear portion of the kinetic curve. As a quality control measure, the Km and kcat of purified SARS-CoV-2 Mpro were compared to published values and only preparations that show similar activity were used in the activity assay. SARS-CoV-2 Mpro was incubated with a test compound for 20 minutes prior to addition of substrate. Compounds were tested at 17 μM in duplicates. For each tested compound, assay results were reported as % fluorescence generated by initial enzyme activity (initial enzyme activity in the absence of test compound). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |