Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris

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 mu g of purified soluble rBoNTF(H-c) were completely protected when challenged with 1000 mouse ip LD50 of BoNTF toxin, Similarly, three doses of 1 mu g of purified resolubilized rBoNTF(H-c) completely protected mice from a challenge of 5000 mouse ip LD50 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, (C) 2000 Academic Press.