Binding of tomatidine with 3CLpro detected by fluorescence quenching assay and ITC. A Expression and purification of recombinant 3CLpro. (i) BL-21 cells transformed with recombinant plasmids pET-32a-3CLpro were subjected to SDS-PAGE. Lane 1, BL21-pET-32a-3CLpro without IPTG induction; Lane 2, BL21-pET-32a with IPTG induction; Lane 3, BL21-pET-32a-3CLpro with IPTG induction; Lane 4, BL21-pET-32a-3CLpro with IPTG induction supernatant after ultrasonication; Lane 5, BL21-pET-32a-3CLpro with IPTG induction precipitation after ultrasonication. (ii) Recombination protein supernatant before (Lane 1) and after (Lane 2) purifications were subjected to SDS-PAGE. (iii) Purified protein was analyzed by Western blot. B Upper: Fluorescence emission spectra of different concentrations of tomatidine in the 3CLpro solutions at 50 mg/L; Lower: Stern–Volmer plot describes the 3CLpro quenching caused by association with quencher. C Representative thermodynamic profiles of tomatidine binding with the 3CLpro in solution, results of ITC measurements.