Expression and Purification of SARS-CoV-2 Main Protease (Nonstructural Protein 5, NSP5) v1
Kaitlyn Varela, Francis K. Yoshimoto · 2023
SARS-CoV-2 is the virus that caused the global COVID-19 pandemic in 2020-2023. This protocol describes how to express and purify the main protease of SARS-CoV-2 (nonstructural protein 5, NSP5) with a C-terminal hexahistidine tag. SARS-CoV-2 main protease is the enzyme (protein) consisting of 306 amino acid residues, responsible for the release of other important proteins in the virus' life cycle (i.e. NSPs 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16), including RNA polymerase (NSP12), which copies the virus' genome in the host. The inhibition of SARS-CoV-2 main protease activity is a strategy to kill the virus. The purified protein in this protocol was used to biochemically characterize SARS-CoV-2 main protease and identify a small molecule inhibitor (i.e. arteannuin B) in a previously published manuscript from our research lab: Varela, K.; Arman, H.D.; Berger, M.S.; Sponsel, V.M.; Lin, C.-H.A.; Yoshimoto, F.K. Inhibition of Cysteine Proteases via Thiol Michael Addition Explains the Anti-SARS-CoV-2 and Bioactive Properties of Arteannuin B. J. Nat. Prod. 2023, 86, 1654-1666. The purified protein was determined to be active through an assay using a model peptide substrate (4-fluoro-FLQS sequence). The peptide product (4-fluoro-FL) was detected using liquid chromatography mass spectrometry. The protease activity was inhibited with arteannuin B, a natural product isolated from the plant, Artemisia annua. Arteannuin B blocked the activity of the enzyme by forming a covalent bond with the active site cysteine residue of SARS-CoV-2 main protease (cysteine-145). Covalent bond formation was determined by mass spectrometry.