Purification, Characterization, and Oral Toxicity of Botulinum Type G Progenitor Toxin

Abstract

Clostridium botulinum type G (or C. argentinense) strain 2470 was grown overnight at 37°C in chopped meat glucose medium and transferred to a medium consisting of proteose peptone 4%, yeast extract 1%, glucose 1%, and L-cysteine 0.2%, pH 7.3, which was incubated for 6 days at 30^°C. The 10-L culture with a potential toxicity of 1.3 x 10⁸ mouse i.p. LD₅₀ was brought to pH 4.0 with sulfuric acid and kept overnight at 4°C. The precipitate was packed by centrifugation and extracted twice with 0.2 M phosphate buffer, pH 6.0. The extract was treated with 0.5% trypsin (Difco 1:250) at 37°C for 60 min. The extract recovered 1.3 × 10⁸ LD₅₀. The residual cells were sonicated and treated with trypsin in a similar way, which recovered 1.1 × 10⁸ LD₅₀. The two extracts were combined, the toxicity of which was set as 100%. Ammonium sulfate was added to the combined extracts to a 0.5 saturation. The precipitate was dissolved in 150 ml of 0.2 M phosphate buffer, pH 6.0, which was clarified by centrifugation. This extract was dialyzed against 0.05 M acetate buffer, pH 4.0, which caused precipitation of the toxin. The precipitate was dissolved in 100 ml of 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. It was clarified by centrifugation, concentrated by salting out, followed by dialysis. The extract of the second salting out recovered 191 mg of protein and 3.3 × 10⁸ LD₅₀ (118%). The extract, divided into 20-ml portions, was subjected to gel filtration on Sephadex G-200 (2.5 × 90 cm). The effluent in the void volume contained 96.3 mg protein and 2.8 × 108 LD50 (100%). The fraction was dialyzed against 0.5 M NaC1-0.05 M acetate buffer, pH 4.0, and added were the same volume of 0.5 M NaCl-0.05 M citrate buffer, pH 4.5, and 0.01% protamine. This was clarified by centrifugation and percolated through SP-Sephadex C-50 (2.5 × 30 cm) equilibrated with 0.5 M NaC1-0.05 M acetate buffer, pH 4.5. The percolate was diluted 2.5-fold with 0.05 M acetate buffer, pH 4.0, applied again to SP-Sephadex C-50 (1.6 × 12 cm) equilibrated with 0.2 M NaC1-0.05 M acetate buffer, pH 4.0, and eluted with NaC1 gradient from 0.2 to 0.7 M. The fractions eluted at 0.34 to 0.48 M NaC1 contained 48.0 mg of protein and 1.9 × 10⁸ LD₅₀ (68%). The toxin fraction was concentrated by salting out and subjected to the second gel filtration on Sephadex G-200 (2.5 × 90 cm) with the same buffer. A single protein peak eluted contained 22.9 mg of protein and 1.1 × 10⁸ LD₅₀ (39%). The specific toxicity was 3.0 × 10⁷ LD₅₀/mg N.