title: Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris.
abstract: A recombinant vaccine candidate was developed that protected mice against botulinum neurotoxin serotype F (BoNTF) intoxication. A synthetic gene encoding BoNTF fragment C (rBoNTF(H(c))) was designed, constructed, and inserted into a plasmid for expression in the yeast Pichia pastoris. A total cell protein content of 2.9 g was obtained per liter of fermentation broth. Recombinant rBoNTF(H(c)) was purified from the soluble yeast extract in two chromatographic steps. The process employed Mono S cation exchange chemistry followed by Alkyl-Superose hydrophobic interaction chromatography, producing material judged to be greater than 98% pure by SDS-PAGE. The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste. rBoNTF(H(c)) was also purified from the insoluble fraction of the yeast cell lysate. Because the fragment C in the pellet was 35% of the total insoluble protein, only a Mono S cation exchange chromatography step was necessary to achieve a purity greater than 98%. Mice that received three injections of 0.2 microgram of purified soluble rBoNTF(H(c)) were completely protected when challenged with 1000 mouse ip LD(50) of BoNTF toxin. Similarly, three doses of 1 microgram of purified resolubilized rBoNTF(H(c)) completely protected mice from a challenge of 5000 mouse ip LD(50) of BoNTF toxin. Individual serum antibody ELISA titers of mice injected with soluble rBoNTF(H(c)) correlated with survival as all 34 mice with ELISA titers of 100 or greater survived toxin challenge. The work presented here demonstrates that purified rBoNTF(H(c)) is able to protect against a high challenge dose of neurotoxin.
authors.affiliation: Department of Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, 21702, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Byrne
authors.forename: R W
authors.initials: RW
authors.lastname: Titball
authors.forename: J
authors.initials: J
authors.lastname: Holley
authors.forename: L A
authors.initials: LA
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000633:toxicity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005285:Fermentation; D007273:Injections, Intramuscular; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D001905:Botulinum Toxins; C096321:botulinum toxin type F