Identification of a novel temperature sensitive promoter in cho cells

BMC Biotechnology

Research Article

Friedemann Hesse¹  Jens Pontiller¹  Haruthai Thaisuchat²  Martina Baumann²  Wolfgang Ernst³ 

[1] Austrian Center of Biopharmaceutical Technology, Muthgasse 18, 1190, Vienna, Austria;Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190, Vienna, Austria;Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 11, 1190, Vienna, Austria;Austrian Center for Industrial Biotechnology (ACIB), A-8010, Graz, Austria;

关键词: Chinese Hamster Ovary Cell;    Chinese Hamster Ovary;    Core Promoter;    Promoter Construct;    S100a6 Gene;   

DOI  :  10.1186/1472-6750-11-51

 received in 2010-07-27, accepted in 2011-05-12,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsBased 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 37°C and 33°C 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.ConclusionsThis novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, 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.

【 授权许可】

Unknown   
© Thaisuchat et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.