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Fig. 2 | Parasites & Vectors

Fig. 2

From: Fowlerstefin, a cysteine protease inhibitor of Naegleria fowleri, induces inflammatory responses in BV-2 microglial cells in vitro

Fig. 2

Production and characterization of recombinant fowlerstefin. a Expression and purification of recombinant fowlerstefin. The recombinant fowlerstefin was purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. Lane 1: non-induced E. coli lysate (20 µg); Lane 2: IPTG-induced E. coli lysate (20 µg); Lane 3: Ni-NTA affinity purified fowlerstefin (10 µg). b Dose-dependent inhibition assay. Inhibitory activity of fowlerstefin against human cathepsin B (HCB), human cathepsin L (HCL), papain and NfCPB-L was analyzed. Each individual enzyme (20 nM) was incubated with different concentrations of fowlerstefin and the residual enzyme activity was measured. The assay buffers for each enzyme were as follows: HCB and HCL, 50 mM sodium acetate (pH 6.0); papain, 50 mM sodium acetate (pH 5.0); and NfCPB-L, 50 mM sodium acetate (pH 4.5). Results are expressed as the percentage of inhibited enzyme activity compared to control without fowlerstefin. Three independent assays were performed for each enzyme and mean and standard deviation (SD) were calculated. c Inhibitory activity of fowlerstefin. Fowlerstefin was incubated with HCL, HCB, papain or NfCPB-L in different pH buffers for 30 min at room temperature, after which the residual enzyme activity of each enzyme was assayed. All experiments were performed in triplicate and the mean and SD values were represented. d Thermal stability. Fowlerstefin was incubated in 50 mM sodium phosphate (pH 7.0) at 37 °C or 95 °C for the indicated times, after which the residual inhibitory activity for HCL, HCB and NfCPB-L was analyzed. All experiments were performed in triplicate and the mean and SD values were calculated

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