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
| Autor: | Ian Henderson, Ardis Barthuli, Jayanta Sinha, Sarah A. Fanders, Mark A. Gouthro, John T. Ross, Michael M. Meagher, Theresa J. Smith, Mehmet Inan, Leonard A. Smith, Rick Barent, Bonnie M. Loveless, Todd Swanson, Shinichi Taoka |
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| Rok vydání: | 2007 |
| Předmět: |
clone (Java method)
Botulinum Toxins Bioengineering Biology Models Biological Applied Microbiology and Biotechnology Pichia Pichia pastoris law.invention Industrial Microbiology chemistry.chemical_compound Bioreactors law Escherichia Escherichia coli Glycerol Computer Simulation Biological Specimen Banks chemistry.chemical_classification General Medicine biology.organism_classification Recombinant Proteins Chemically defined medium Enzyme Biochemistry chemistry Bacterial Vaccines Recombinant DNA Fermentation Biotechnology |
| Zdroj: | Journal of Biotechnology. 127:462-474 |
| ISSN: | 0168-1656 |
| Popis: | A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) 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(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) 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(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis. |
| Databáze: | OpenAIRE |
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