Fig. 1. SDS–PAGE (10%) analysis of rLF protein expressed in E. coli. (A) The effect of induction time on GST-rLF expression. Lanes 1 to 4, respectively, represented the induction time of 2 h (lane 1), 4 h (lane 2), 6 h (lane 3) and 8 h (lane 4) with 0.2 mM IPTG at 28 °C. The expected molecular weight of GST-rLF was about 116 kDa. (B) Identification of GST-free rLF production. lane 1, 3 C protease-treated rLF purified by GSH affinity chromatography; lane 2, clarified supernatant of cell lysate containing GST-rLF; lane 3, insoluble fraction without GST-rLF; lane 4, clarified supernatant of the lysate of cells carrying pGEX-6P-1 vector after IPTG induction as negative control; lane M, reference proteins as the markers of molecular weight.

Fig. 2. Cytotoxicity of purified rLF and the mutant rLFm-Y236F on RAW264.7 cells. Cells grown in 96-well plates were treated with various concentrations of rLF and rLFm-Y236F along with 1 μg/ml of rPA. After 3 h of incubation, cell viability was determined by using alamarBlue staining.

Fig. 3. Immunological assay of rLF. (A) Western blot assay of rLF. Lane 1, the purified rLF; lane 2, supernatant of cell lysate containing the blank vector as negative control. (B) Neutralization of anthrax toxin by mouse anti-LF antibody. Various dilutions of anti-LF serum were pre-incubated with rLF (■) for 1 h. The mixture was then added to RAW264.7 cells along with 1 μg/ml of rPA for 5 h. The cell viability was determined by using alamarBlue staining. (), positive control: the cells 1 μg/ml rLF and 1 μg/ml rPA; (□), Negative control: the cells without rLF and PA.