Lys6, Lys11, Lys29, Lys33, Lys48, Lys63, and linear linked di-Ub obtained from Boston Biochem were incubated at 10 μM with 20 nM BtSCoV-Rf1.2004 PLpro. Reactions were performed in AMC buffer at a volume of 45 μL and a temperature of 37°C. 10 μL samples were taken at the indicated time points and heat-shocked at 98°C for 5 min. Lys48 and Lys63 linked tetra-Ub obtained from Boston Biochem were incubated at 13.67 μM with 23 nM BtSCoV-Rf1.2004 PLpro. Reactions were performed in AMC buffer at a volume of 70 μL and a temperature of 37°C. 8 μL samples were taken at the indicated time points and heat-shocked at 98°C for 5 min. SDS-PAGE analysis was performed using Mini-PROTEAN TGX and Coomassie blue.
Freitas B.T., Ahiadorme D.A., Bagul R.S., Durie I.A., Ghosh S., Hill J., Kramer N.E., Murray J., O’Boyle B.M., Onobun E., Pirrone M.G., Shepard J.D., Enos S., Subedi Y.P., Upadhyaya K., Tripp R.A., Cummings B.S., Crich D, & Pegan S.D. (2022). Exploring noncovalent protease inhibitors for the treatment of SARS and SARS-like coronaviruses. ACS infectious diseases, 8(3), 596-611.
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