High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D_HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D_HCR was obtained. The recombinant BoNT/D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.