Expression, purification, and efficacy of the type A botulinum neurotoxin catalytic domain fused to two translocation domain variants

Toxicon. 2003 May;41(6):691-701. doi: 10.1016/s0041-0101(03)00042-4.

Abstract

Clostridial neurotoxins are potent inhibitors of synaptic function, with the zinc-dependent proteolytic light chain (LC) portion of the toxin cleaving one of three neural SNARE proteins. In nature, the LC is expressed as a part of a much larger toxin and hemagglutinin complex, protecting it from environmental degradation and preserving its catalytic activity. We developed forms of the LC of type A botulinum neurotoxin (BoNT-A) with parts of the larger toxin gene, for use as reagents in high-throughput assays to screen for potential LC antagonists, to further elucidate the toxin's mechanism of action, and to study immunological responses to the toxin. Three BoNT-A constructs were engineered and expressed: the LC, LC with translocation region (LC+H(n)), and the LC with the belt portion of the translocation region (LC+Belt). Purification was optimized to a two-step process, with relatively high yields of all three constructs obtained. Activity assays showed all three constructs to be active, with the LC being the most active. Immunogenic protection against native BoNT-A toxin challenge was observed for all three constructs, with the best protection observed with the LC+H(n) and LC+Belt proteins.

MeSH terms

  • Animals
  • Botulinum Toxins, Type A / genetics
  • Botulinum Toxins, Type A / immunology
  • Botulinum Toxins, Type A / isolation & purification*
  • Botulinum Toxins, Type A / metabolism*
  • Catalytic Domain*
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression*
  • Genetic Engineering*
  • Mice
  • Protein Transport
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Temperature

Substances

  • Recombinant Fusion Proteins
  • Botulinum Toxins, Type A