Rapid method for purification of Clostridium botulinuh type C neurotoxin by High Performance Liquid Chromatography (HPLC )

Abstract

The culture supernatant of Clostridium botulinum type C, concentrated by addition of RNA, acid precipitation and subsequent protamine treatment was used as starting material for rapid purification of L toxin (mol. wt. ca. 500K) and M toxin (mol. wt. ca. 350K) of C1 neurotoxin by ion-exchange chromatography on a Mono S column by fast performance liquid chromatography (FPLC). L and M toxins were highly purified further by gel permeation chromatography through a TSK G3000SW column at pH 6.0 by high performance liquid chromatography (HPLC). Purified S toxin (mol. wt. ca. 150K, Cl neurotoxin without a nontoxic component) was then obtained from L toxin rapidly by gel permeation chromatography at pH 7.3 through a TSK G3000SW column by HPLC. Purified S toxin was also obtained rapidly from M and L toxins by ion-exchange chromatography on a Mono Q column at pH 8.0 using an FPLC system. The purified preparations of L, M and S toxins gave single bands on conventional polyacrylamide gel electrophoresis, and had specific activities of 2.8, 6.7, and 14–21 × 10⁷ LD₅₀/mg N, respectively, in mice. On immunoelectrophoresis, purified S toxin gave a single arc against anti-crude toxin serum. The yield of toxicity as L and M toxins was 73.1% (32.5% as L toxin and 40.6% as M toxin) from the protamine-treated concentrated culture supernatant. The recovery of toxicity as S toxin from purified L or M toxin was almost 100% (97.6–100% of L toxin and 97.5% of M toxin). These procedures provide a rapid method for purifying L and M toxins, which have stable toxicities. The method will also be very useful for rapid preparation of the toxic component (S toxin) of C1 neurotoxin, which is unstable, in small amounts from the L and M toxins just before its use in experiments.