Production and Purification of the Heavy Chain Fragment C of Botulinum Neurotoxin, Serotype A, Expressed in the Methylotrophic Yeast Pichia pastoris

Potter, Karen J.; Zhang, Wenhui; Smith, Leonard A.; Meagher, Michael M.

doi:10.1006/prep.2000.1256

Cited by 46 publications

(40 citation statements)

References 40 publications

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“…Limited quantities of a pentavalent toxoid vaccine granted Investigational New Drug status in 1979 are available for individuals at risk of exposure. However, due to the difficulties and risks associated with producing toxoid vaccines [25] subsequent efforts have largely focused on recombinant vaccines for prophylaxis [26][27][28][29]. We have previously demonstrated that recombinant rBoNT Hc vaccines are highly efficacious, protecting against challenges of over 100,000 LD 50 of toxin, often after a single vaccination [30].…”

Section: Discussionmentioning

confidence: 99%

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

Webb¹,

Smith²,

Wright³

et al. 2007

Vaccine

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Recombinant botulinum Hc (rBoNT Hc) vaccines for serotypes C1 and D were produced in the yeast Pichia pastoris and used to determine protection against four distinct BoNT C and D toxin subtypes. Mice were vaccinated with rBoNT/C1 Hc, rBoNT/D Hc, or with a combination of both vaccines and challenged with BoNT C1, D, C/D, or D/C toxin. Mice receiving monovalent vaccinations were partially or completely protected against homologous toxin and not protected against heterologous toxin. Bivalent vaccine candidates completely survived challenges from all toxins except D/C toxin. These results indicate the recombinant C1 and D Hc vaccines are not only effective in a monovalent formula but offer complete protection against both parental and C/D mosaic toxin and partial protection against D/C mosaic toxin when delivered as a bivalent vaccine.

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Section: Discussionmentioning

confidence: 99%

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

Webb¹,

Smith²,

Wright³

et al. 2007

Vaccine

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“…Typically, the methanol induction phase was 10-70 h depending on the antigen, yielding a final cell mass of approximately 60 g dry cell weight per liter of fermentation broth. Fermentation conditions have been worked out for optimum yield of H C for each of the serotypes [69,71,72].…”

Section: Design Of Synthetic Bont(h C ) Gene Construct and Expressimentioning

confidence: 99%

“…At the time of this review, a bioprocess development was completed for BoNT(H C ) serotypes A, B, C 1 , and F [69,[71][72][73][74] (Smith L.A., Byrne M.P., in preparation) and a manufacturing process for serotype E is in development. Purification streams for the vaccines are briefly discussed below and outlined in figure 2.…”

Section: Process For Purifying Bont(h C ) From Pichia Pastorismentioning

confidence: 99%

Development of vaccines for prevention of botulism

2000

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-Botulism is a potentially lethal disease caused by one of seven homologous neurotoxic proteins usually produced by the bacterium, Clostridium botulinum. This neuromuscular disorder occurs through an exquisite series of molecular events, ultimately ending with the arrest of acetylcholine release and hence, flaccid paralysis. The development of vaccines that protect against botulism dates back to the 1940s. Currently, a pentavalent vaccine that protects against BoNT serotypes A-E and a separate monovalent vaccine that protects against BoNT serotype F are available as Investigational New Drugs. However, due to the numerous shortcomings associated with the toxoid vaccines, several groups have efforts towards developing next-generation vaccines. Identifying a synthetic peptide that harbors a neutralizing epitope is one approach to a BoNT vaccine, while another employs the use of a Venezuelan equine encephalitis virus replicon vector to produce protective antigens in vivo against BoNT. The strategy used in our laboratory is to design synthetic genes encoding non-toxic, carboxy-terminal fragments of the C. botulinum neurotoxins (rBoNT(H C )). The gene products are expressed in the yeast, Pichia pastoris, and purified to greater than 98% with yields typically ranging from 200-500 mg per kg of wet cells. Protective immunity to the purified products against high-level challenges of neurotoxin is elicited in mice and in non-human primates. A pre-Investigational New Drug meeting was held with the Food and Drug Administration, and the next milestone for the vaccine candidates will be clinical trials. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS botulinum neurotoxin / vaccine / C-fragment / purification / efficacy

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“…The heavy chain (H c ) fragments of BoNT (A-F) were shown to be non-toxic, antigenic [11] and capable of eliciting a protective immunity in vaccinated animals challenged with homologous BoNT [12]. These results prompted an effort to develop a recombinant botulinum (rBoNT) vaccine against all seven serotypes using the H c fragments as vaccine antigens [10,[13][14][15][16][17]. The H c fragments from each of the seven serotypes [rBoNT(H c )] are distinctly different proteins with varying degrees of homology.…”

Section: Introductionmentioning

confidence: 99%

“…The H c fragments from each of the seven serotypes [rBoNT(H c )] are distinctly different proteins with varying degrees of homology. Due to the differences in the characteristics of each protein and the level of expression of each in Pichia pastoris, a standardized fermentation and purification process was not feasible, resulting in the development of separate processes for rBoNTA(H c ), rBoNTB(H c ), rBoNTE(H c ) and rBoNTF(H c ) [14,[16][17][18] and now rBoNTC(H c ). Serotypes D and G are still under development.…”

Section: Introductionmentioning

confidence: 99%

Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris

Dux

Huang

Barent

et al. 2011

Protein Expression and Purification

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Production and Purification of the Heavy Chain Fragment C of Botulinum Neurotoxin, Serotype A, Expressed in the Methylotrophic Yeast Pichia pastoris

Cited by 46 publications

References 40 publications

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

Development of vaccines for prevention of botulism

Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris

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