Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active

J Protein Chem. 2000 Aug;19(6):475-87. doi: 10.1023/a:1026549431380.


Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4 degrees C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency kcat/Km was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Botulinum Toxins, Type A / antagonists & inhibitors
  • Botulinum Toxins, Type A / genetics
  • Botulinum Toxins, Type A / metabolism*
  • Buffers
  • Catalysis / drug effects
  • Cysteine / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / genetics
  • Exocytosis / drug effects
  • Hydrogen-Ion Concentration
  • Inclusion Bodies / enzymology
  • Inclusion Bodies / genetics
  • Kinetics
  • Metals / pharmacology
  • Mice
  • Mice, Inbred ICR
  • Molecular Sequence Data
  • Pliability
  • Salts
  • Sulfhydryl Reagents / pharmacology
  • Transfection


  • Buffers
  • Metals
  • Salts
  • Sulfhydryl Reagents
  • Botulinum Toxins, Type A
  • Cysteine