Abstract
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.
Highlights
The Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases
Identification of the CHO S100a6 gene Potential promoter candidates were selected from a list of abundantly transcribed genes that was obtained from CHO microarray expression data [14]
The S100a6 was selected as target gene due to its high amount of mRNA levels and its response to “cold shock” conditions in various CHO parental and recombinant cell lines
Summary
The Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. For manufacturing complex biopharmaceuticals the Chinese hamster ovary (CHO) expression system is the leading production platform due to appropriate product secretion, post-translational processing, a simple means of cultivation and up-scaling, a reasonable safety profile and last but not least the approval from regulatory authorities [1,3]. To keep pace with the needs of the market, optimizing efforts are required, including otherwise induce stress responses leading to incorrect protein folding or even apoptosis. Another problem relates to cell-cycle dependence of these promoters that may cause high cell to cell variation in the amount of recombinant protein expressed. More recently the rat ferritin heavy chain (HC) gene locus has been reported by Prentice et al [9] to be suitable for foreign gene expression
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