Figure 1. Schematic of the recombinant proteins used as vaccines and/or antigens to assess the host immune response to vaccination.

(Upper panel) BoNT-derivatives used in this study are shown. His₆ and Strep epitopes were used for protein purification, while 3X-FLAG and two sequential hemagglutinin, 2HA, epitopes were included for cellular studies. Domain junctions were defined, using the crystal structure of BoNT/A1 (PDB:3BTA). Single amino acid designations indicate amino acid substitutions used to reduce catalysis (LC) or receptor binding (HC_C). Note, single chain BoNT and LCHC_N were used for vaccination. (Lower panel) Four μg of the indicated proteins were subjected to SDS-PAGE and Coomassie blue staining. Lanes: 1, M-BoNT/A1; 2, M-BoNT/A1 trypsin nicked and reduced; 3, M-LCHC_N/A1; 4. M-LCHC_N/A1 trypsin nicked and reduced; 5, LC/A1^RY; 6, HC_C/A1^W; and 7, TeNT^RY. Migration of molecular weight marker proteins (kDa) are shown in left lane. Note, in lane 2 nicked HC runs at ~ 80 kDa, which other experiments showed was due to cleavage of the belt region of HC by trypsin.