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Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter
Author(s) -
James Rohaizah I.,
Elton John P.,
Todd Paul,
Kompala Dhinakar S.
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(20000120)67:2<134::aid-bit2>3.0.co;2-v
Subject(s) - mouse mammary tumor virus , chinese hamster ovary cell , microbiology and biotechnology , reporter gene , biology , transfection , cell culture , expression vector , glucocorticoid receptor , gene expression , gene , recombinant dna , biochemistry , genetics
The mouse mammary tumor virus (MMTV) promoter is induced by the addition of a glucocorticoid hormone or analog such as dexamethasone. The hormone binds to its specific transcription factor, glucocorticoid receptor (GR), and the activated complex then binds to the glucocorticoid response elements (GREs) in the enhancer region of the MMTV promoter to induce the overexpression of downstream genes. We have constructed an expression vector for a reporter protein, secreted alkaline phosphatase (SEAP),controlled by the MMTV promoter and co‐transfected this vector along with a GR expression cassette into Chinese hamster ovary (CHO) cells. High producers were cloned and grown in suspension cultures. A very high titer, over 0.4 mg/mL, of SEAP was obtained from this inducible overexpression system, about ten times that achievable with the same reporter protein using the strong constitutive SV40 promoter in CHO cells. A peak production rate of 187 pg SEAP per cell per day was observed within 3 days after induction, compared to the peak rate of 23 pg SEAP per cell per day expressed using the constitutive SV40 promoter. With the reduced or zero growth rate during the protein production phase, this novel MMTV overexpression system is highly suited for optimizing glycoprotein synthesis rates in high cell density fed‐batch or perfusion bioreactors. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 134–140, 2000.