Production and purification of the heavy-chain fragment C of botulinum neurotoxin, serotype B, expressed in the methylotrophic yeast Pichia pastoris

A recombinant H-c fragment of botulinum neurotoxin, serotype B (rBoNTB(H-c)), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 gn (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure H-c fragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified H-c fragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin. (C) 1998 Academic Press.