TRANSFORMATION OF A NONENZYMATIC TOXIN INTO A TOXOID BY GENETIC-ENGINEERING

Curaremimetic toxins are typical non-enzymatic toxins that bind to the ir target [the nicotinic acetylcholine receptor (AChR)] through multip le residues, Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a tox in sufficed to cause an affinity decrease of the toxin for AChR that i s higher than four orders of magnitude, Despite these triple mutations , the overall conformation of the mutated protein remains similar to t hat of a related recombinant toxin, as judged from both circular dichr oism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies, Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wi ld-type toxin to AChR. Therefore, transformation of a non-enzymatic to xin into a toroid can be achieved, like in the case of enzymatic toxin s, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.