Production and Purification of the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype B, Expressed in the Methylotrophic YeastPichia pastoris |
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| Authors: | Karen J Potter Mark A Bevins Elena V Vassilieva Vijay R Chiruvolu Theresa Smith Leonard A Smith Michael M Meagher |
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| Institution: | aBiological Process Development Facility, Department of Food Science and Technology;dDepartment of Biological Systems Engineering, University of Nebraska–Lincoln, Lincoln, Nebraska, 68583-0919;bDepartment of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania, 16802;cToxicology Division, U.S. Army Medical Research Institute for Infectious Disease (USAMRIID), Fort Detrick, Frederick, Maryland, 21702-5012 |
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| Abstract: | A recombinant Hcfragment of botulinum neurotoxin, serotype B (rBoNTB(Hc)), has been successfully expressed in a Mut+strain of the methylotrophic yeastPichia pastorisfor 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 Hcfragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified Hcfragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin. |
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