Purification of Clostridial Neurotoxins

TeTx is isolated from culture supernatants of Clostridium tetani by ammonium sulfate precipitation followed by chromatography (Mat-suda and Yoneda, 1975 Schiavo and Montecucco, 1995). Single chain TeTx is released by bacterial lysis and is rapidly converted by bacterial proteases into the di-chain TeTx form (Kriegistein et ai, 1991 Das-Gupta, 1994). Different growth conditions and extraction procedures 

BoNTs can be isolated from bacterial culture by acid precipitation and citrate extraction of the pellet, followed by ammonium sulfate fractionation and chromatography on SP-Sephadex (Shone and Tranter, 1995). Alternatively, CNTs can also be obtained from different commercial sources (see 14.8). 

Protease-free preparation of CNTs can be obtained using an immobilized-metal-ion affinity chromatography (IMAC) step (Ros-setto et a/., 1992). This procedure is also useful for the purification of He, the 50 kDa carboxy-terminal part of the heavy chain of TeTx, which shows an identical retention time. However, IMAC-chromatography cannot be used for the purification of BoNT/D because this serotype is not retained. To obtain a protease-free BoNT/D preparation, an ion-exchange chromatography procedure was used (Schiavo and Montecucco, 1995). After freezing in liquid nitrogen, purified CNTs are stored at -80°C. 

Toxicity is routinely tested by intraperitoneal injection of different amounts of toxin. Mouse LD50 is 0.1-1 ng/kg (Payling-Wright, 1955 Gill, 1982). Toxicity of CNTs varies in different animal species (Gill, 1982), and this is due to different binding at the neuromuscular junction and/or to mutations at the site of action inside cells. 

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