Expression and purification of the light chain of botulinum neurotoxin A: a single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain.

Botulinum neurotoxin type A (BoNT/A) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings. While the toxin's heavy (H) chain contributes to neuronal binding and internalization, its light (L) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNAP-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by Tyr. The PCR-amplified wild-type and mutant L chain genes were cloned, fused to the gene for maltose-binding protein, and expressed at high levels in Escherichia coli. The soluble fusion proteins were purified using amylose affinity chromatography, and, after factor Xa cleavage, the free L chains were isolated. The wild-type was shown to proteolyze SNAP-25 at a rate approaching that of the native chain while the mutant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo. After reconstitution with the H chain, the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays. This methodology allows, for the first time, routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and, thereby, assist the design of effective inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)