Identification of a novel temperature sensitive promoter in cho cells

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The <it>S100a6 </it>(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.</p> <p>Conclusions</p> <p>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.</p

Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Exploiting Nucleotide Composition to Engineer Promoters

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications

Plasmids and plasmid amounts used for various experiments.

<p>pEGFP-N1, pcDNA luc, pRC-LC and pRC-HC carry the CMV immediate-early promoter, pGL3-control and pRL carry SV40 promoter elements, pTripolis and pBi-NP drive bidirectional transcription (mRNA and vRNA) of the influenza polymerases and the NP protein. pMono-EGFP, pMono-lucNS, pMono-LC, pMono-HC, pMono-lucM and pMono-lucPB1 drive monodirectional (vRNA) transcription of the gene of interest. AB stands for antibody, LC stands for light chain experiments.</p

Schematic drawings of plasmids used to generate the influenza replicon based expression system.

<p>pTripolis and pBi-NP generate the influenza replicon (mRNA/protein and vRNA) whereas pMono plasmids with various genes of interest or reporter genes drive transcription of vRNA.</p

Influence of different NCRs on the expression level

<p>. HEK-293 cells were tranfected with the influenza replicon and either pMono-lucNS (NCR NS) or pMono-lucM (NCR M) or pMono-lucPB1 (NCR PB1) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for <i>Renilla</i> luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have been normalized to <i>Renilla</i> values. Data represents arithmetic mean values and standard deviation.</p

FACS analysis of cells transfected with either pEGFP-N1 (CMV) or the influenza replicon system and pMono-EGFP (Flu Replicon) 77 hours post transfection.

<p>Cells transfected with the replicon system show two very distinct populations in the forward light scatter (EGFP), one completely negative, the other highly positive. pEGFP-N1 driven EGFP expressions shows a broad distribution spanning from completely negative to moderate and high expressing cells.</p