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

Abstract

A recombinant Hc fragment of botulinum neurotoxin, serotype B (rBoNTB(Hc)), 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 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure Hc fragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified Hc fragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.