Comparative Analysis of MAR and Terminator Elements in Controlling Stable EPO Expression in CHO-K1 Cells

Abstract

Purpose: Modulating chromatin organization and post-transcriptional regulation are critical for improving recombinant protein production . The human β-globin MAR (β-MAR) enhances transgene expression through nuclear matrix tethering, whereas the β-globin terminator (β-TERM) is implicated in transcriptional termination . However, their combined effects, particularly in stable erythropoietin (EPO) expression in CHO-K1 cells, remain less understood. Methods: A codon-optimized EPO sequence were cloned into four expression plasmids. Stable polyclonal CHO-K1 cell pools were generated and assessed for transgene copy number, steady-state mRNA abundance, and EPO secretion . In parallel, in silico RNA-binding protein (RBP) profiling was performed for the polyA signal and β-TERM, followed by protein–protein interaction network analysis, hub-gene identification, and functional enrichment to elucidate post-transcriptional regulatory mechanisms. Results: Incorporation of β-MAR resulted in remarkable EPO expression, yielding approximately 15 gene copies per cell, a 5.3-fold increase in mRNA abundance, and an EPO titer of 28.3 mIU/mL (3.4-fold over control). In contrast, the β-TERM element alone exerted minimal effects on protein output, while the combined β-MAR+β-TERM construct showed intermediate performance, suggesting partial attenuation of β-MAR activity. Network analysis of RBPs associated with poly(A) and β-TERM sequences revealed a highly interconnected post-transcriptional regulatory network enriched in RNA processing and stability factors. Notably, HNRNPK, HNRNPA2B1, U2AF2, FUS, and CELF1 were identified as central hub genes, indicating that β-TERM predominantly influences RNA fate rather than directly enhancing translation. Conclusion: These findings establish β-MAR as a dominant chromatin-level enhancer of stable EPO expression in CHO cells and demonstrate that β-TERM engages RBP-centered post-transcriptional networks in a context-dependent and non-additive manner. This integrative analysis provides mechanistic insight for the rational design of expression cassettes in mammalian cell engineering and biopharmaceutical production.

Keywords: MAR, Stable cell line, Erythropoietin, Terminator, CHOk1