Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Repression of toxin production by tryptophan in Clostridium botulinum type E

Of the seven amino acids required by Clostridium botulinum type E, tryptophan is the most essential and may provide the cell with nitrogen. The addition of excess tryptophan (10–20 mM) or other nitrogenous nutrients to minimal growth medium markedly decreased toxin formation but did not affect growth in C. botulinum type E. On the other hand, the addition of an enzymatic digest of casein (NZ Case) stimulated toxin formation and overcame repression by tryptophan. Immunoblots of proteins in culture fluids using antibodies to type E toxin indicated that tryptophan-repressed cultures produced less neurotoxin protein. Inhibitors of neurotoxin did not accumulate in cultures grown in minimal medium supplemented with high tryptophan. The results suggest that tryptophan availability in foods or in the intestine may be important for toxin formation by C. botulinum type E.     

Activation of Clostridium botulinum type E toxin purified by two different procedures

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.     

Monoclonal antibody to type F Clostridium botulinum toxin

Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was approximately 10 mouse lethal doses of toxin per ml of toxic fluid.     

Immunochemical study of Clostridium botulinum type B toxin

Fractionation of type B. Cl. botulinum toxin, a protein complex, was carried out; as a result, 3 fractions, alpha, beta, and gamma, were isolated in a pure form, alpha-fraction, or neurotoxin, is highly toxic (5-10.10(7) LDm per 1 mg of protein), beta-fraction showed hemagglutinating activity (64-128 HAU per 1 mg of protein), gamma-fraction was not biologically active. The molecular weight of alpha and gamma-fractions was 150,000. All these fractions had antigenic properties. alpha-fraction was serologically specific. beta- and gamma-fractions showed incomplete serologic identity.     

A new scaleable method for the purification of botulinum neurotoxin type E

Botulinum neurotoxins belong to the most toxic substances in nature. Well-known as a food poisoning agent for almost two centuries, the beneficial aspects of this bacterial metabolite were rediscovered about 30 years ago. These toxins, which are produced by the anaerobic bacterium Clostridium botulinum are nowadays used to treat a variety of neuro-muscular disorders. The increased demand requires techniques for the production and purification of these toxins on an industrial scale. The method described herein is based on filtration and chromatography procedures only. Precipitation, centrifugation and dialysis steps were consequently excluded to develop a protocol, which can easily be scaled up from the laboratory purification to industrial needs. About 4 mg of BoNT/E were purified from a 10-L batch culture corresponding to an overall recovery of approximately 14%.