Scale-Up of the Fermentation and Purification of the Recombinant Heavy Chain Fragment C Of Botulinum Neurotoxin Serotype F, Expressed in Pichia pastoris

Authors

• Scott K. Johnson, University of Nebraska - LincolnFollow
• Wenhui Zhang, University of Nebraska - Lincoln
• Leonard A. Smith, Division of Toxinology and Aerobiology, United States Army Medical, Research Institute of Infectious Diseases, Fort Detrick, Fredrick, MDFollow
• Karen J. Hywood-Potter, University of Nebraska - Lincoln
• S. Todd Swanson, University of Nebraska - Lincoln
• Vicki L. Schlegel, University of Nebraska-LincolnFollow
• Michael M. Meagher, Department of Chemical Engineering, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2003

Comments

Published in Protein Expression and Purification 32 (2003) 1–9.

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 fedbatch 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 10–100mL 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.