COMPARATIVE PRODUCTION AND CHARACTERIZATION OF A CATALYTICALLY INACTIVE BoNT/C1 HOLOPROTEIN IN TWO PRODUCTION STRAINS OF PICHIA PASTORIS
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Hood College Biology
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Biomedcial and Environmental Science
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Abstract
Botulinum neurotoxins (BoNTs) are the causative agents of the disease botulism.
At present no FDA-licensed vaccine exists for the prevention of BoNT intoxication.
Efforts to produce a catalytically inactive BoNT/CI holoprotein (ciBoNT/CI HP) vaccine
in Pichia pastoris X-33 by a government contractor were hampered by extensive
proteolysis of the recombinant protein. In an effort to reduce proteolysis, the ciBoNT/CI
HP was expressed and purified from both X-33 and the protease knockout PichiaPink.
By modifying the fermentation method and purification protocol, the level of proteolytic
degradation was significantly improved over that obtained by the contractor. However,
equivalent levels of ciBoNT/CI HP proteolysis were observed from both X-33 and
PichiaPink. The purified vaccine was found to be both safe and protective in mice with a
median effective dose of 13.6 ng (X-33) and 14.7 ng (PichiaPink) in a single-dose mouse
potency assay involving challenge with 1,000 MLD50 of BoNT/C.