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Titel
Identification of a novel temperature sensitive promoter in cho cells
VerfasserThaisuchat, Haruthai ; Baumann, Martina ; Pontiller, Jens ; Hesse, Friedemann ; Ernst, Wolfgang
Erschienen in
BMC Biotechnology, 2011, Jg. 11,
ErschienenBioMed Central (BMC), 2011
SpracheEnglisch
DokumenttypAufsatz in einer Zeitschrift
ISSN1472-6750
URNurn:nbn:at:at-ubbw:3-694 Persistent Identifier (URN)
DOIdoi:10.1186/1472-6750-11-51 
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 Das Werk ist frei verfügbar
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Identification of a novel temperature sensitive promoter in cho cells [0.4 mb]
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Zusammenfassung (Englisch)

Background:

The Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature.

Results:

Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin) and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37C and 33C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp) comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence.

Conclusions:

This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33C, a two to three-fold increase of basal productivity (already higher than SV40 promoter) is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.