Cloning, expression and purification of botulinum neurotoxin type A heavy chain - crystallographic evidence for a putative tetrameric pore

Clostridium botulinum produces seven antigenically distinct serotypes of botulinum neurotoxin (BoNTs, A-G) the most potent toxins to humans.They cause paralysis at less than picomolar concentration by blocking neurotransmitter release.BoNT consists of a heavy chain and a light chain.The C-terminal half of the heavy chain allows the toxin to bind to the presynaptic membrane while the N-terminal half helps in forming a channel in the endosomal membrane to allow the light chain to escape into the cytosol.While the binding and catalytic mechanisms are well understood now, the details of translocation still remain a mystery.A full length BoNT/A heavy chain (BAHC) has been cloned, over expressed and purified from inclusion bodies by solubilising with a detergent.Preliminary crystallographic results show that BAHC forms a tetramer in the crystal lending experimental support for tetrameric pore formation for the light chain to pass through the endosomal membrane.