Production and Purification of the Heavy Chain Fragment C of Botulinum Neurotoxin, Serotype A, Expressed in the Methylotrophic Yeast Pichia pastoris

Authors

• Karen J. Potter, University of Nebraska - Lincoln
• Wenhui Zhang, University of Nebraska - Lincoln
• Leonard A. Smith, University of Nebraska - LincolnFollow
• Michael M. Meagher, Department of Chemical Engineering, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2000

Comments

Published in Protein Expression and Purification 19, 393-402 (2000).

Abstract

A recombinant Hc fragment of botulinum neurotoxin, serotype A (rBoNTA(Hc)), has been successfully expressed in a Mutt strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(H_c) production while minimizing proteolytic degradation. Concentration of rBoNTA(H_c) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The H_c fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The H_c fragment was purified to >95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified H_c fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin.