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 inhi bits acetylcholine release from peripheral nerve endings. While the to xin's heavy (H) chain contributes to neuronal binding and internalizat ion, its light (L) chain is a Zn2+-dependent endoprotease that intrace llularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNA P-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His(227) in the Zn2+-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 X(a) cleavag e, the free L chains were isolated. The wild-type was shown to proteol yze SNAP-25 at a rate approaching that of the native chain while the m utant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form tha t inhibited neuromuscular transmission in vitro and produced the sympt oms of botulism in vivo. After reconstitution with the H chain, the Ty r(227) mutant L chain failed to show any neuroparalytic activity in ei ther of these assays. This methodology allows, for the first time, rou tine preparation of recombinant forms of the L chain that are needed t o decipher the molecular details of its interaction with substrate and , thereby, assist the design of effective inhibitors. Moreover, the ge neration herein of a nontoxic dichain that retains ability to bind, in ternalize, and translocate to the cytosol of motor nerve terminals, by reconstituting inactive L chain with its native partner, could provid e a targeted vehicle for the transport of drugs into peripheral cholin ergic neurons.