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Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system

Par Conseil national de recherches du Canada

DOITrouver le DOI : https://doi.org/10.1016/j.jbiotec.2008.04.006
AuteurRechercher : 1; Rechercher : ; Rechercher : ; Rechercher : 1; Rechercher : 1; Rechercher : ; Rechercher :
Affiliation
  1. Conseil national de recherches du Canada. Institut de recherche en biotechnologie du CNRC
FormatTexte, Article
SujetCMV promoter; BacMam baculovirus; hollow fiber (HBFR); factorial design; Sf9; Chinese hamster ovary (CHO-K1); HEK 293A cells; wave bioreactor; TFF, tangential flow filtration; human embryonic kidney cells (HEK); secreted alkaline phosphatase (SEAP); trichostatin; alkaline phosphatase; biotechnology; cell Line; cell; cytomegalovirus; gene expression; in vitro; kidney; phosphatase; protein; vitro
Résumé
A BacMam baculovirus was designed in our laboratory to express the reporter protein secreted alkaline phosphatase (SEAP) driven by the immediate early promoter of human cytomegalovirus promoter (CMV). In vitro tests have been carried out using this recombinant baculovirus to study the secreted protein in two cell lines and under various culture conditions. The transductionswere carried out on two commonly used mammalian cell lines namely the human embryonic kidney (HEK 293A) and Chinese hamster ovary (CHO-K1). Initial studies clearly demonstrated that the transient expression of SEAP was at least 10-fold higher in the HEK 293 cells than the CHO cells under equivalent experimental conditions. Factorial design experiments were done to study the effect of different parameters such as cell density, MOI, and the histone deacetylase inhibitor, trichostatin A concentration. The multiplicity of infection (MOI) and the cell density were found to have the most impact on the process. The enhancer trichostatin A also showed some positive effect. The production of secreted protein in a batch reactor was studied using the Wave disposable bioreactor system. A semi-continuous perfusion process was developed to extend the period of gene expression in mammalian cells using a hollow fiber bioreactor system (HFBR). The growth of cells and viability in both systems was monitored by offline analyses of metabolites. The expression of recombinant protein could be maintained over an extended period of time up to 30 days in the HFBR.
Date de publication
Dans
  • Journal of Biotechnology 135, nº June 3 (17 avril 2008) : 272–280.
NoteDA - 20080623IS - 0168-1656 (Print)LA - engPT - Journal ArticleSB - IM
Langueanglais
Publications évaluées par des pairsOui
Numéro du CNRC47825
NRCC 47825
Numéro NPARC12400994
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Identificateur de l’enregistremente4f6f7f0-0bb1-4da0-816e-0eb008a7b663
Enregistrement créé2009-10-02
Enregistrement modifié2020-04-15
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