Production and purification of the heavy-chain fragment C of botulinum neurotoxin, serotype B, expressed in the methylotrophic yeast Pichia pastoris.
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.
Department of Food Science and Technology, Biological Process Development Facility, University of Nebraska-Lincoln, 68583-0919, USA.
K J
KJ
Potter
M A
MA
Bevins
E V
EV
Vassilieva
V R
VR
Chiruvolu
T
T
Smith
L A
LA
Smith
M M
MM
Meagher
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D009498:Neurotoxins; D011994:Recombinant Proteins; D001905:Botulinum Toxins