Recombinant Botulinum Toxoids: A Practical Guide for Production
Clostridium botulinum is a Gram-positive, spore-forming, anaerobic bacillus that produces a potent neurotoxin. Botulinum neurotoxins (BoNTs) are classified from serotypes A to H, and even though they have similar mechanisms of action, they show preferenti
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Introduction Botulism is a disease caused by toxins produced by Clostridium botulinum [1]. This Gram-positive, rod-shaped bacterium is a spore-forming, strict anaerobe that produces a potent neurotoxin classified from serotypes A to H [2]. Despite this classification, botulinum neurotoxins (BoNTs) have similar mechanisms of action, causing severe and often fatal flaccid muscle paralysis due to the inhibition of acetylcholine release into neuromuscular junctions [3]. In animals, BoNT serotypes C and D are the most important, affecting most of the farm animals [4]. All BoNTs are synthesized as a single polypeptide that is cleaved by C. botulinum proteases and then connected again by a disulfide bound. Two chains and three domains compose the structure of a 150 kDa active BoNT: the light chain (LC), which comprises the catalytic domain, and the heavy chain (HC), which comprises the translocation and binding domains. LC is a 50 kDa metalloprotease that is connected to the HC by the disulfide bound, while HC is divided in two 50 kDa regions: the N-terminal region (HN), which is the translocation domain, and the C-terminal region (HC), which is the binding domain [5, 6]. Several studies have shown that HC alone is nontoxic [1, 7]. Furthermore, considering the fact that this region is capable of generating high levels of protection when used as vaccine antigen, most of the studies focus on the use of HC [8–10]. The conventional method for the production of vaccines against botulism involves the culture of C. botulinum, and further purification and inactivation of the neurotoxins [1, 11]. Since this microorganism is a strict anaerobe and requires special components on its
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_40, © Springer Science+Business Media New York 2016
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culture medium, the production process becomes complex [12]. Moreover, C. botulinum fermentation involves high biosafety levels, offering intoxication risks to workers. Another drawback is that C. botulinum strains show variable yield of toxin production between different batches, causing a problem for the industry. The long time for toxin inactivation (approximately 10 days) and the presence of residual formaldehyde (the most commonly used inactivation agent) in the vaccine are also disavantages of the conventional process. The production of recombinant vaccines using Escherichia coli seems to be the best option to circumvent the shortcomings of the current methods. E. coli expression system allows the use of simple media that result in a reproducible high-yield production. Biosafety risks are also not as big as those for C. botulinum, since E. coli strains used for heterologous expression are not pathogenic and will be used to produce a nontoxic region of the toxin, which dispenses the use of inactivation components such as formaldehyde [7]. Considering this, we describe a method for t
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