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.

Today the standard technology for the production of many biopharmaceutical products from mammalian cell systems is frequently based on expression vectors that utilize strong mammalian active viral promoters like CMV or SV40 for driving recombinant gene transcription. On one hand these promoters allow very high expression rates, but on the other hand, they can lead to constitutive over-expression of the gene of interest resulting in a permanent stress on the cell. Another drawback is that they are cell cycle-dependent and can be subject to gene silencing which leads to a heterogeneity within the cell population. Here we aim to identify endogenous gene regulatory elements that are capable of controlling the transcription of the foreign gene. For this purpose, a genomic CHO library containing fragments of various lengths was constructed using a shotgun cloning strategy. After enzymatic fragmentation of genomic DNA, fragments encoding potential promoter regions were inserted into a promoterless vector upstream of the neomycin resistance gene. This random pool of library plasmids was transfected back into CHO cells and via cell sorting directly into 96-well plates and G418 (neomycin) selection, positive clones were isolated. A nested PCR of resistant CHO clones resulted in potential fragments which were sequenced. Putative promoter activity was predicted for these sequences by in silico methods and will be proved by re-transfection of reporter constructs into CHO cells.