Recombinant SARS-CoV-2 3CLpro shows a concentration-dependent increase in net A625/A525 ratio which is dependent upon 3CLpro activity.
(A) 1 µg untagged, purified recombinant SARS-CoV-2 wild-type (WT), H41A and H163A 3CLpro protein was separated by SDS–PAGE and total protein visualised with Coomassie Blue stain; (B) Recombinant SARS-CoV-2 3CLpro shows a concentration dependent increase in net A625/A525 ratio (error bars represent means ± standard deviation; n = 3 independent experiments); (C) Compared with untreated (blue points) samples, treatment with 50 nM (orange points) and 500 nM (grey points) GC376 significantly reduces recombinant SARS-CoV-2 3CLpro concentration-dependent increases in net A625/A525 ratio (error bars represent means ± standard deviation; n = 3 independent experiments; **** P < 0.0001, * P < 0.05 by two-way ANOVA Dunnett's Multiple Comparisons Test compared with Untreated sample); (D) Recombinant SARS-CoV-2 H41A and H163A 3CLpro mutants and wild-type denatured (WT DN) recombinant SARS-CoV-2 3CLpro show no significant increase in net A625/A525 ratio above background levels (−WT) compared with wild-type (WT) SARS-CoV-2 3CLpro (error bars represent means ± standard deviation; n = 3 independent experiments; **** P < 0.0001 by one-way ANOVA Dunnett's Multiple Comparisons Test). A representative visual image from independent replicate experiments is shown in a panel below the graph.