Mitigating transcriptional bottleneck using a constitutively active transcription factor, VP16-CREB, in mammalian cells

Abstract

High-level expression of recombinant proteins in mammalian cells has long been an area of interest. Inefficient transcription machinery is often an obstacle in achieving high-level expression of recombinant proteins in mammalian cells. Synthetic promoters have been developed to improve the transcription efficiency, but have achieved limited success due to the limited availability of transcription factors (TFs). Here, we present a TF-engineering approach to mitigate the transcriptional bottlenecks of recombinant proteins. This includes: (i) identification of cAMP response element binding protein (CREB) as a candidate TF by searching for TFs enriched in the cytomegalovirus (CMV) promoter-driven high-producing recombinant Chinese hamster ovary (rCHO) cell lines via transcriptome analysis, (ii) confirmation of transcriptional limitation of active CREB in rCHO cell lines, and (iii) direct activation of the transgene promoter by expressing constitutively active CREB at non-cytotoxic levels in rCHO cell lines. With the expression of constitutively active VP16-CREB, the production of therapeutic proteins, such as monoclonal antibody and etanercept, in CMV promoter-driven rCHO cell lines was increased up to 3.9-fold. VP16-CREB was also used successfully with synthetic promoters containing cAMP response elements. Taken together, this strategy to introduce constitutively active TFs into cells is a useful means of over-coming the transcriptional limitations in recombinant mammalian cells.