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Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris.

Abstract：

:A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Potter KJ,Zhang W,Smith LA,Meagher MM

doi

10.1006/prep.2000.1256

keywords:

subject

Has Abstract

pub_date

2000-08-01 00:00:00

pages

393-402

issue

3

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(00)91256-7

journal_volume

19

pub_type

杂志文章

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