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

Paper ID Volume ID Publish Year Pages File Format Full-Text
25031 43553 2008 9 PDF Available
Title
Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system
Abstract

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 transductions were 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.

Keywords
HEK, human embryonic kidney cells; SEAP, secreted alkaline phosphatase; CHO, Chinese hamster ovary cells; IU, infectious units, or international units; ICS, intra-capillary space; ECS, extra capillary space; FBS, fetal bovine serum; MOI, multiplicity of i
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Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 135, Issue 3, 30 June 2008, Pages 272–280
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