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. 2011 May 12:11:51.
doi: 10.1186/1472-6750-11-51.

Identification of a novel temperature sensitive promoter in CHO cells

Haruthai Thaisuchat  1 , Martina Baumann, Jens Pontiller, Friedemann Hesse, Wolfgang Ernst
Affiliations

Identification of a novel temperature sensitive promoter in CHO cells

Haruthai Thaisuchat et al. BMC Biotechnol. .

Abstract

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 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.

Conclusions: This 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.

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Figures

Figure 1
Figure 1
Map of the CHO S100a6 gene. The exon intron architecture and structural details of the CHO S100a6 gene are illustrated. Depicted sequence spans from -1505 bp of the 5' untranslated region to the end of exon 3 at +650 bp. Predicted transcriptional start site (TSS) is located in exon 1 at position -620 (arrow). Translation initiates in exon 2 after the first 24 bp at position +1. The predicted CpG island overlaps with exon 1 and extends from nucleotides -553 to -742 (dotted line). Putative transcription factor binding sites (TFBS) around TSS and in intron 1 are indicated in the figure.
Figure 2
Figure 2
Reporter constructs of the CHO S100a6 gene promoter. The full-length construct (Sps1) encodes 1505 nts upstream the ATG. Grey boxes at the top indicate nts-numbers. Sps-constructs comprise the entire promoter region starting with nt 1 (unsigned) and are truncated at the 5'-side. 3' truncations (dS) start with nt 910 and are successively shortened at their 3' terminus. For double truncations (p3, p4), deletion (16-∆SP1), core promoter (dS18), tandem core promoter (18doub) and the reverse-orientated constructs see text. TFBS are indicated with boxes, TSS is marked with an arrow.
Figure 3
Figure 3
Normalized luciferase activity of reporter constructs of the S100a6 promoter. After co-transfections cells were incubated at 37°C and 33°C and measured for lucifearse activity after 24 hours. The average of two independent experiments with triplicate samples is shown for each construct. Relative promoter activity is indicated on the y-axis, as a percentage value of the SV40 control at the respective temperature.
Figure 4
Figure 4
Regulatory elements in the S100a6 promoter region. Alignment among mouse, rat and Chinese hamster (CHO). NF-ĸB, SP1 binding sites and TATA box are conserved sequences and indicated by boxes.
Figure 5
Figure 5
Luciferase activity of S100a6 promoter constructs. 5 different constructs of the S100a6 promoter in series 4 compared to dS16, +C (pGL3-P, the positive control) and -C (pGL3-B, the negative control). Each construct was co-transfected with pRL-SV40 into CHO cells. Cell suspensions were measured for luciferase activity 24 hours after transfection and incubation at 33°C. All relative luciferase activities were normalized to Renilla luciferase activity. The average of two independent experiments with triplicate samples is shown for each construct.
Figure 6
Figure 6
Temperature-shift induced change in gene expression. Two representative promoter constructs, the Sps1 full-length and the dS18 core promoter sequence, were analyzed for relative mRNA levels (qRT) and luciferase protein expression (Luc). Analyses were performed at two different temperatures at 33°C and 37°C measured after 24 hours. The shown values represent the mRNA and protein ratios at 33°C relative to 37°C (y-axis) normalized to pGL3 control vector.
Figure 7
Figure 7
mRNA stability of cold shock transcripts. The full-length promoter construct (Sps1, 1505 nts) was analyzed by quantitative RT-PCR after inhibition of de novo transcription in order to determine transcript stability at 33°C and 37°C. The y-axis shows the percent-values of the amount of mRNA of the Luc reporter construct at indicated time points (hours after transfection). Time zero is set to 100%. After 6 hrs at 37°C the mRNA decline was 33% as compared to 5% at 33°C. The columns show the ratio of transcript abundance (33°C-value is set to 100%) that was detected at 24 hrs after transfection at both temperatures.
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