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

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Sinha J., Inan M., Fanders S., Taoka S., Gouthro M., Swanson T., ...Daha Fazla

JOURNAL OF BIOTECHNOLOGY, cilt.127, sa.3, ss.462-474, 2007 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 127 Sayı: 3
  • Basım Tarihi: 2007
  • Doi Numarası: 10.1016/j.jbiotec.2006.07.022
  • Dergi Adı: JOURNAL OF BIOTECHNOLOGY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.462-474
  • Anahtar Kelimeler: recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, Pichia pastoris, cell-bank characterization, fed-batch fermentation, potency studies, HIGH-LEVEL EXPRESSION, OVINE INTERFERON-TAU, CLOSTRIDIAL NEUROTOXINS, METHYLOTROPHIC YEAST, TETANUS TOXIN, VACCINES, SNAP-25, PROTEIN, EXOCYTOSIS, METHANOL
  • Akdeniz Üniversitesi Adresli: Hayır

Özet

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. (c) 2006 Elsevier B.V. All rights reserved.