terminator hinders the higher erythropoietin production achieved through Matrix Attachment Region in CHO-K1 cells

Strategies to achieve substantial yield from Chinese hamster ovarian (CHO) cell factories remain crucial in the biopharmaceutical industry. Chromosome remodeling, mRNA 3' end extension diversity, mRNA maturation, and the utilization of upstream and downstream DNA elements have been shown to impact the production of recombinant proteins significantly. In this study, we employed the human beta-globin Matrix Attachment Region (β-MAR) as an upstream element and the human beta-globin terminator (β-TERM) as a downstream section to augment erythropoietin (EPO) production. EPO is a crucial medicine in treating certain forms of anemia produced through recombinant DNA technology. In this experiment, four plasmids expressing EPO transgene, after codon bias and secondary structure of EPO mRNA optimization, were transfected into CHO-K1 cells. Two weeks after antibiotic selection, pooled stable clones were evaluated for gene copy number (GCN), mRNA quantification, and EPO expression level. Our results revealed a significant increase in the amount of EPO secreted by stable CHO cells in the presence of β-MAR, while β-TERM had no noticeable impact on EPO production. As expected, the presence of the β-MAR sequence resulted in a higher gene copy number with 15 gene copy per cell (GCN/cell) and mRNA quantity with 5.3 fold change, leading to a much greater secretion of EPO with 28.3 mIU/ml compared to the β-TERM sequence with 3 GCN/cell, 2.8 mRNA fold change and 8.2 mIU/ml of EPO quantity. This experiment further supports previous studies, demonstrating the significant impact of the β-MAR element on recombinant protein production in stable CHO-K1 cells.