Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development. [Display omitted] •CHO-K1 endogenous promoters were identified based on RNA-Seq data of high-density cell cultures•Activity of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 promoters was compared to CMV + Enhancer•The newly discovered promoters exhibited high-level transgene expression in CHO, BHK and HEK cells•Stable expression following a reduction of the culture temperature was investigated•These promoters showed outstanding results and may help to avoid well-known drawbacks of viral-derived promoters