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 P2(1)2(1)2(1), 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.
Cell Biology and Biochemistry Group, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Yanfeng
Y
Zhang
Xiaoli
X
Gao
Ling
L
Qin
Garry W
GW
Buchko
Howard
H
Robinson
Susan M
SM
Varnum
D015153:Blotting, Western; D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D003014:Clostridium botulinum / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
C096325:botulinum toxin type D; D001905:Botulinum Toxins