Nsp5 expression, purification and activity test by nsp9 gel-based assay.
(A) Schematic overview of nsp5 purification. Crude cell extracts containing His14-SUMO tagged nsp5 is bound to a Ni-NTA resin, washed with ATP and then eluted by His14-SUMO cleavage by the protease Ulp1. Major contaminates are then removed over an anion exchange and gel filtration column. (B) Coomassie gel of His14-SUMO cleavage. Panel 1: Elution from Ni-NTA beads before and after cleavage of nsp5 by the Ulp1 SUMO-dependent protease. Panel 2: Sample of the flow through from the anion exchange column. Panel 3: Input for gel filtration column. Panel 4: Fractions taken across the major peak of the gel filtration elution. Lanes six through eight (in bold) were pooled and concentrated for use in the HTS screen. (C) Schematic overview of nsp9 gel-based cleavage assay. The substrate of FLAG-His-SAVLQ-NNEL…VRLQ, where NNEL…VRLQ represents full length nsp9, is incubated nsp5. Cleavage of the substrate results in two products: nsp9 and FLAG-His-SAVLQ. (D) Gel-based assay for nsp9 cleavage by nsp5 over time at 500 and 1000 nM of nsp5. Nsp5 is visible at ∼35 kDa and the substrate of uncleaved nsp9 visible at ∼12 kDa.