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Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(Hc): Antigen E) by Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
25486 43576 2007 13 PDF Available
Title
Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(Hc): Antigen E) by Pichia pastoris
Abstract

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(Hc) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(Hc) gene inserted into pHILD4 Escherichia coli—P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(Hc) sequence. Expression of rBoNTE(Hc) from the Mut+HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(Hc). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(Hc) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(Hc) per gram wet cell mass as determined by HPLC and Western blot analysis.

Keywords
Recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E; Pichia pastoris; Cell-bank characterization; Fed-batch fermentation; Potency studies
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Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(Hc): Antigen E) by Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 127, Issue 3, 10 January 2007, Pages 462–474
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us