The production of rBoNT/A1 (32) and rBoNT/B1 (25) has been described previously. In summary, constructs were cloned in the pET32a expression vector and mutations introduced by site-directed mutagenesis. Recombinant molecules were expressed in E. coli in selective media using 1 mM isopropyl-β-D-thiogalactopyranoside at 16°C for 20 hours. Cells were harvested and lysed, and the lysate was clarified before purification of target protein by fast protein liquid chromatography with BuHP Sepharose (GE Healthcare Life Sciences) and Q Sepharose High Performance (GE Healthcare Life Sciences). The partially purified molecule was then cleaved with Lys-C to yield the active di-chain, which was polished down a phenyl Sepharose High Performance (GE Healthcare Life Sciences) column. All recombinant toxins were purified and activated to more than 85%, as determined by densitometry, and molecule identity was confirmed by SDS–polyacrylamide gel electrophoresis (PAGE) and Western blot analysis (fig. S6, A and B).

The production of Hc/B was performed, as described previously (15). Briefly, the codon-optimized gene was cloned into the pET28a expression vector and expressed as above. Hc/B was purified by immobilized metal affinity chromatography with HisTrap HP (GE Healthcare Life Sciences), followed by gel filtration with Superdex 200 (GE Healthcare Life Sciences). The sample was kept in 20 mM Hepes (pH 7.5), 300 mM NaCl, 10% glycerol, and 0.5 mM TCEP [tris(2-carboxyethyl)phosphine].

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