Monolight 3010

We tested the ability of recombinant PvNod22 to inhibit client protein aggregation at a high temperature by measuring the recovery activity with a luciferase activity assay and SDS-PAGE gels. First, 1 µM luciferase (Promega, Madison, WI, USA) was incubated for 20 min at 42 °C in the absence or presence of either 1 or 3 µM PvNod22 in 25 mM HEPES/KOH (pH 7), 10 mM KCl, 5 mM MgCl₂ and 2 mM dithiothreitol. Then, the luciferase was incubated in a solution containing 30 μL of nuclease-treated rabbit reticulocyte lysate at 30 °C to refold the protein. The luciferase recovery activity was measured at different times (5 to 90 min) in a luminometer (Monolight 3010, BD Biosciences, San Jose, CA, USA). The activity of the unheated sample was used as a control. Data points and associated error bars represent three independent PvNod22 purifications. Additionally, either 5 or 1.5 µM of luciferase were suspended in 20 μL of PBS pH 7.5 and incubated at 42 °C in the absence or presence of 40 µM PvNod22 (1:8 and 1:26 molar ratios of luciferase: PvNod22, respectively) for 20 min. Then, the solution was incubated for 1.5 h at 30 °C, and protein aggregates were recovered by centrifugation (16,000× g for 5 min). The proteins present in the supernatants and pellets were analyzed by 15% (w/v) SDS-PAGE.