Fig. 2. Purification of TeNT, L chain of TeNT, Hc of TeNT, BoNT/A, and BoNT/D by HPLC. (A) Chelating Superose-Zn(II) chromatography: samples were eluted with the imidazole gradient shown in the top graph, and the retention time for each peak is indicated. (B) BoNT/D was eluted from a Mono Q HR HPLC column with the NaCl step gradient depicted as a dashed line. (C) The SDS-PAGE profiles of BoNT/D before chromatography (U), peak 1 (neurotoxin), and peak 2 (nontoxic peptide). BoNT serotypes A, B, and E, obtained as in (A), are also shown.

chain is loaded onto a zinc-IMAC column to eliminate residual TeNT. The L chain is collected in the void volume, dialyzed against 20 mM Tris-Cl, pH 8.0, and applied to a Mono Q HR 5/5 column previously equilibrated with the same buffer. The L chain is eluted with a linear gradient of NaCl (0-200 mM) at a 1.0 ml/min flow rate. Fractions containing the purified protein are collected and precipitated with ammonium sulfate (60% saturation). The resulting L chain is homogeneous on both SDS-PAGE and reversed-phase chromatography on a BU-300 column (100 X 46 mm; Pierce, Rockford, IL). The L chain is kept at 4°; alternatively, after freezing in liquid nitrogen, it is stored at -80° as indicated above.

Zinc Content Determination. Routinely the zinc content and the animal toxicity of the purified toxins are measured as a quality control of the

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