| Assay Method Information | |
| | Enzyme Assays and Inhibition Studies |
| Description: | Cloning and Expression of the 3CL Protease of SARS-CoV-2 and FRET Enzyme Assays. The codon-optimized cDNA of full length of 3CLpro of SARS-CoV-2 (GenBank number MN908947.3) fused with sequences encoding six histidines at the N-terminal was synthesized by Integrated DNA (Coralville, IA). The synthesized gene was subcloned into the pET-28a(+) vector. The expression and purification of SARS-CoV-2 3CLpro were conducted following a standard procedure described previously. Briefly, a stock solution of an inhibitor was prepared in DMSO and diluted in assay buffer composed of 20 mM HEPES buffer, pH 8, containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol. The SARS-CoV-2 protease was mixed with serial dilutions of the inhibitor or with DMSO in 25 μL of assay buffer and incubated at 37 °C for 1 h, followed by the addition of 25 μL of assay buffer containing the substrate (FAM-SAVLQ/SG-QXL520, AnaSpec, Fremont, CA). The substrate was derived from the cleavage sites on the viral polyproteins of SARS-CoV. Fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) for 1 h following the addition of the substrate. Relative fluorescence units were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously.25 The dose-dependent FRET inhibition curves were fitted with a variable slope using GraphPad Prism software (GraphPad, La Jolla, CA) in order to determine the IC50 values of the compounds. The expression and purification of the 3CLpro of MERS-CoV and the FRET enzyme assays were performed as described previously. |
| Affinity data for this assay | |
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