Identification of a novel temperature sensitive promoter in cho cells

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.

Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells

“…Thus, specific enhancement of the target gene transcription at 32°C by using MCRE is a promising method to increase the final yield of recombinant proteins in various cell lines. Interestingly, Thaisuchat et al [21] have recently identified a novel temperature sensitive promoter (222 bp long) in the S100a6 (calcyclin) gene, which comprises two Sp1 sites.…”

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

“…The other approach is to regulate the ST6Gal I expression. Introduction of regulatory expression system into CHO cell, i.e., Tet-on/off system (Chung et al 2004) and the temperature-sensitive promoter (Thaisuchat et al 2011), would be effective in minimizing the disadvantage of ST6Gal I overexpression. In addition, a recent report indicated that therapeutic antibodies produced by CHO cells often contain the non-human sialic acid, N-glycolylneuraminic acid (Neu5Gc), to which Neu5Gc-specific human antibodies show antigenicity (Ghaderi et al 2010).…”

Identification of a novel endogenous regulatory element in Chinese hamster ovary cells by promoter trap

“…However, this approach is very labor-intensive and can yield potentially confounding results. Other methods include screening the flanking regions of highly expressed genes obtained from CHO microarray data for potential promoter candidates; this approach, however, limits identification to known genes (Thaisuchat et al, 2011).…”