Fig. 6: Trigger factor eliminates non-productive oligomerization and facilitates Vibrio harveyi luciferase refolding.
From: Trigger factor both holds and folds its client proteins
a and b Sedimentation velocity-analytical ultracentrifugation analysis of a 2 μM refolding luciferase and b 2 μM refolding luciferase in the presence of 8-fold molar excess (16 μM) of trigger factor at 4 °C shows that the multiple species present for refolding GAPDH are resolved to a single TF–GAPDH complex in the presence of excess trigger factor. All samples were cooled down to 4 °C in the centrifuge over a period of 3 h, until the temperature equilibrated at 4 °C. All experiments were performed in buffer A containing 0.1 M urea, 4 °C. Data were analysed by two-dimensional sedimentation analysis, followed by a genetic algorithm-Monte Carlo analysis. c Fluorescence anisotropy binding curves of fluorescently labelling trigger factor upon titration with urea-denatured luciferase. The titration experiment was performed in buffer A at 25 °C. The data points were fitted to a hyperbola equation to determine the dissociation constant (Kd). Each anisotropy measurement is the average of 20 independent measurements, and the error reported is the standard deviation. Value of Kd reported is the mean ± s.e.m. of the fit. d In vitro bacterial luciferase activity assay. 1.06 μM refolding luciferase was incubated with various concentrations of trigger factor (0–10.6 μM) at 25 °C. The enzymatic activity of refolding luciferase was monitored at various refolding time points. Each data point is the average of three independent measurements, and the error reported is the standard deviation.
