Endogenous eukaryotic CHO-K1 cell promoters as tools to improve the production of a difficult-to-express recombinant protein

Recombinant proteins with applications in human and veterinary medicine are mainly produced in mammalian expression systems, particularly Chinese hamster ovary (CHO) cells. Although these cells enable proper folding and post-translational modifications, they often exhibit low specific productivity, which represents a critical limitation in large-scale biopharmaceutical manufacturing. Strong viral promoters, including CMV and its enhancer-enriched variant CMV+E, are commonly used to drive transgene expression. However, they are susceptible to epigenetic silencing and progressive loss of activity during prolonged culture, which can compromise yield stability. Endogenous host-cell promoters constitute a promising alternative, as they can maintain transgene expression in alignment with physiological and bioprocess regulatory mechanisms. Here, we assessed the activity of endogenous eukaryotic promoters previously identified in our laboratory from highly expressed CHO-K1 genes. The receptor-binding domain (RBD) of SARS-CoV-2 was selected as a model of a complex secretory glycoprotein domain. Our results demonstrate that the Hspa5 and Vim promoters enable sustained and high-level expression of recombinant RBD protein in CHO-K1 cells, achieving performance comparable to the enhancer-containing viral promoter CMV+E. Notably, Hspa5 and Vim are unoptimized genomic sequences lacking classical enhancers; nevertheless, their intrinsic stability and reduced susceptibility to silencing make them attractive regulatory elements for recombinant protein production. These findings highlight their potential for next-generation promoter engineering and for the development of long-term, high-yield mammalian expression systems for complex therapeutic proteins.