Figure 2.

Construction and characterization of an anthrax–plague triple antigen. (A) Schematic of protective antigen (PA) and F1mutV-PA recombinant constructs. The PA20 domain of PA is shown in yellow, the PA63 domain is shown in blue, PA domain IV is shown in red, F1mut is shown in green, and V is shown in purple. Furin cleavage site and its cleavage products are also indicated. (B) Structural model of F1mutV-PA triple antigen. The model was manually generated using Chimera with structures of F1 (PDB ID: 1Z9S), V (PDB ID: 4JBU), and PA (PDB ID: 1ACC). (C) Binding to PA receptor, CMG2. The purified PA or F1mutV-PA proteins were incubated with increasing amounts of CMG2 and interactions between the PA proteins and CMG2 were analyzed by native-PAGE. PA-CMG2 and F1mutV-PA-CMG2 complexes are marked with red arrows. PA and F1mutV-PA are marked with black arrows. (D) Furin cleavage. The PA or F1mutV-PA proteins were treated with increasing amounts of furin and the cleavage products were analyzed by SDS-PAGE. Positions of the PA and F1mutV-PA bands are marked with red arrows and the positions of the cleaved products PA63, PA20, and F1mutV-PA20 bands are marked with blue arrows. (E) Binding to N-terminal domain of lethal factor (LFn). The PA and F1mutV-PA proteins were first treated with furin to release PA63 and then incubated with increasing amounts of LFn. Interactions between the PA63 heptamer/octamer and LFn were analyzed by native-PAGE. The PA63–LFn complexes are marked with braces. The SDS-PAGE and native gels were stained with Coomassie blue R-250 and Coomassie blue G-250, respectively.