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 HC fragment) is a promising vaccine candidate. Among the HC fragments of the seven BoNT serotypes, the expression of HC/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 HC/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 HC/B expression levels were obtained when the bacterium E. coli BL21(DE3)+pET-9a-HC/B was grown in terrific broth media at 18°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 HC/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 HC/B was highly immunogenic, protecting mice from a 10(6) LD50 challenge after a single vaccination and generating a neutralizing titer of 50IU/ml 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.
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Alon
A
Ben David
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Amram
A
Torgeman
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
Ada
A
Barnea
Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel. Electronic address: ranz@iibr.gov.il.
Ran
R
Zichel
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D019274:Botulinum Toxins, Type A / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001906:Botulism / Q000276:immunology / Q000382:microbiology / Q000401:mortality / Q000517:prevention & control*; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016019:Survival Analysis; D014611:Vaccination
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C096323:rimabotulinumtoxinB; D006639:Histidine; D019274:Botulinum Toxins, Type A; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase
Botulinum vaccine; His-tag protein purification; Recombinant protein expression