Expression, purification and characterization of the receptor-binding domain of botulinum neurotoxin serotype B as a vaccine candidate

The receptor-binding domain of botulinum neurotoxins (the H-C fragment) is a promising vaccine candidate. Among the H-C fragments of the seven BoNT serotypes, the expression of H-C/B in Escherichia coli is considered especially challenging due to its accumulation as a non-soluble protein aggregate. In this study, the effects of different parameters on the expression of soluble H-C/B were evaluated using a screening assay that included growing the bacterium at a small scale, a chemical cell lysis step, and a specific ELISA. The highest soluble H-C/B expression levels were obtained when the bacterium E. coli BL21(DE3) + pET-9a-H-C/B was grown in terrific broth media at 18 degrees C without induction. Under these conditions, the yield was an order of magnitude higher than previously reported. Standard purification of the protein using a nickel column resulted in a low purity of H-C/B. However, the addition of an acidic wash step prior to protein elution released a major protein contaminant and significantly increased the purity level. Mass spectrometry analysis identified the contaminant as ArnA, an E. coli protein that often contaminates recombinant His-tagged protein preparations. The purified H-C/B was highly immunogenic, protecting mice from a 10(6) LD50 challenge after a single vaccination and generating a neutralizing titer of 50 Mimi after three immunizations. Moreover, the functionality of the protein was preserved, as it inhibited BoNT/B intoxication in vivo, presumably due to blockade of the neurotoxin protein receptor synaptotagmin. (C) 2015 Elsevier Inc. All rights reserved.