Scale-up of the fermentation and purification of the recombinant heavy chain fragment C of botulinum neurotoxin serotype F, expressed in Pichia pastoris

Scott K. Johnson, Wenhui Zhang, Leonard A. Smith, Karen J. Hywood-Potter, S. Todd Swanson, Vicki L. Schlegel, Michael M. Meagher

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

A recombinant heavy chain fragment C of botulinum neurotoxin serotype F (BoNTF(Hc)) has been expressed in Pichia pastoris for use as an antigen in a proposed human vaccine. P. pastoris cells were grown using glycerol batch, glycerol fed-batch, and methanol fed-batch methods to achieve high cell densities. The total cellular protein recovered after homogenization was 72 mg/ g of cell paste. BoNTF(Hc) was purified from soluble Pichia cell lysate employing ion-exchange chromatographic (IEC) and hydrophobic interaction chromatographic (HIC) methods developed at the bench scale using 10-100 mL columns. The process was performed at the pilot scale using 1-4 L columns for evaluation of scale up. The purification process resulted in greater than 98% pure product consisting of at least three forms of BoNTF(Hc) based on mass spectrometry and yielded up to 205 mg/kg cells at the bench scale and 170 mg/kg cells at the pilot scale. Full-length BoNTF(Hc) is present based on mass spectrometry and SDS-PAGE, however is postulated to be N-terminally blocked by acetylation. N-terminal sequencing showed that two of the three forms are missing the first 11 (80%) and 14 (20%) amino acids of the N-terminus from the full-length form. The ratios of the two clipped forms were consistent from the bench to pilot scales. Purified BoNTF(Hc) at the pilot scale was found to sufficiently protect mice against a high dose of BoNTF neurotoxin.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalProtein Expression and Purification
Volume32
Issue number1
DOIs
StatePublished - Nov 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology

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