Recombinant firefly luciferase

Confirmed qHTS active compounds were tested in a biochemical assay to directly measure potential for luciferase inhibition using Kinase-Glo (Promega). Briefly, 3 μl of substrate buffer (50 mM Tris–HCl [pH 7.5], 10 mM MgCl₂, 0.05% bovine serum albumin, 0.01% Tween-20, 13.33 μM d-luciferin (Sigma; catalog no.: L9504), and 13.33 μM ATP) were dispensed into 1536-well assay plates. Compounds were then transferred via Kalypsys pin tool equipped with 1536-pin array. Following addition of compound, 1 μl of recombinant firefly luciferase (Sigma; L9506 prepared in 50 mM Tris–HCl [pH 7.5], 10 mM MgCl₂, 0.05% bovine serum albumin, and 0.01% Tween-20; 10 nM final) was added to initiate the reaction. After 10 min of room temperature incubation, end-point measurements of luminescence were acquired using a ViewLux plate reader equipped with clear filters.

Heat induced aggregation of 300 nm recombinant firefly luciferase (Sigma‐Aldrich, St Louis, MO, USA) in buffer G (50 mm HEPES/KOH, pH 7.5, 100 mm KCl, 10 mm MgCl₂, 1 mm EDTA, 1 mm DTT and 10% (v/v) glycerol) was monitored by UV absorbance at 320 nm for 25 min at 43 °C in a Varian Cary Eclipse fluorescence spectrophotometer (Santa Clara, CA, USA) equipped with a temperature controller. Various concentrations of ClpB or ClpB∆N protein were added to the reaction, with 5 mm of ATP or other indicated nucleotides, prior to heat aggregation and their effect on the aggregation of luciferase was recorded. The end‐point value of the absorbance of aggregated luciferase alone was considered as 100% aggregation. The percentage aggregation of other variables was calculated relative to this value.

Recombinant firefly luciferase (Sigma-Aldrich, St. Louis, Missouri, United States) was diluted to 10⁴ U/mg in 1× PBS (phosphate buffered saline) and subsequently incubated with NFA or the substances of interest for 15 min at 37°C. Afterwards, 50 µl per reaction mix was transferred into a white 96-well plate, to which 50 µl/well Dual-Glo® (Promega, Madison, Wisconsin, United States) was added and incubated for another 10 min at 37°C. Luminescence was analyzed with a plate reader.