Study design and protein preparation
Active TeNT and decoy TTc R1225E W1288A and LCHn were expressed from the pRSET vector constructs in E. coli BL21 (DE3). The decoy molecules comprised the binding domain of tetanus toxin (TTc) with two point-mutations to prevent neuronal binding and a separately produced enzymatic-translocation domain (LCHn). The mutations introduced in TTc were reported to be involved in neuronal binding (Sutton et al. , 2001, Bakry et al. , 1991, Chen et al. , 2009, Ovespianet al. , 2015, Qazi et al. , 2006), and LCHn was inactive due to the peptide bond between the light-chain (LC) and Hn domain and absence of a binding domain (TTc) (Figure 1A, B and C). The overall flow of the animal experiments is represented schematically in figure 1D, and an example of tetany stage observations is shown in figure 1E.
TeNT and decoy proteins were produced in milligram amounts, per litre of culture, at purity levels estimated to be greater than 95% (Figures 2A, B and C). Post-purification, full-length TeNT was cleaved into its active di-chain form by treatment with TPCK-trypsin. The activated protein, when analysed by SDS-PAGE, showed the expected pattern of separate 100 kDa and 50 kDa protein subunits representing the heavy and light chains of the toxin, respectively (Figure 2A). The presence of several different sizes of light-chain is expected due to slight variations in cleavage by trypsin. This has been reported previously and does not appear to affect the activity of the protein (Ahnert-Hilgeret al. , 1990, KRIEGLSTEIN et al. , 1991). The presence of the disulphide bond between the heavy- and light-chains, required for the biological activity of the toxin, was confirmed by comparing the protein on reducing and non-reducing SDS-PAGE (Figure 2A). The presence of a single 150 kDa protein on the non-reducing gel confirmed the presence of the disulphide bond. Even though E. coli BL21 is reported to have a reducing cytosol, the protein was produced with an oxidised disulfide bond. Whether the bond was formed during protein folding withing the cell, or spontaneously during the purification or activation process is unknown. Identity of the proteins were confirmed by Western blot using primary antibodies derived against tetanus toxoid (Rabbit anti-TeNT IgG). Polyclonal antibodies recognised both the TTc and LCHn components of the decoy (Figure 2B and C), suggesting that both subunits are targeted by the immune response. This is consistent with studies investigating TeNT immunity (da Silva Antunes et al. , 2017, Lavinder et al. , 2014, Palermo et al. , 2017).