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

Journal Article · Wed Dec 01 04:00:00 EST 2010 · Acta Crystallographica. Section F, 66(12):1610-1613

DOI:https://doi.org/10.1107/S1744309110039874· OSTI ID:1000139

Zhang, Yanfeng; Gao, Xiaoli; Qin, Lin; Buchko, Garry W; Robinson, Howard; Varnum, Susan M

Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level with high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1000139
• Report Number(s): PNNL-SA-74543; 34699; 41092; 400412000
• Journal Information: Acta Crystallographica. Section F, 66(12):1610-1613, Journal Name: Acta Crystallographica. Section F, 66(12):1610-1613 Journal Issue: 12 Vol. 66
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
BIOLOGY
Botulism
CHAINS
CRYSTALLIZATION
DATA ANALYSIS
DIMENSIONS
DISEASES
Environmental Molecular Sciences Laboratory
L CELLS
PRODUCTION
PROTEINS
PURIFICATION
RESOLUTION
SOLUBILITY
SPACE GROUPS
VACCINES
X-ray protein crystallography
structural biology