Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein

Abstract The importance of anti-apoptosis in mammalian cell culture has been widely recognized. We reported previously that expression of Bombyx mori 30 K genes in Chinese hamster ovary (CHO) cells increases recombinant protein production by inhibiting apoptosis and enhancing specific productivity. However, previous studies have shown expression of the anti-apoptotic 30Kc6 protein as inclusion bodies in Escherichia coli. 30Kc19 protein, another silkworm hemolymph protein, has cell-penetrating and recombinant protein productivity-improving properties, and we found that it improves soluble expression of its partner. In this study, we fused 30Kc6 with 30Kc19 as an expression partner to express a soluble fused protein and to deliver the protein to cells. Supplementing the recombinant 30Kc19-30Kc6 fusion protein in cell culture medium increased viability, effectively penetrated the cells, and inhibited CHO cell apoptosis by 68%. Moreover, the mitochondrial membrane potential and ATP generation also increased by 50% and 33%, respectively. Erythropoietin (EPO) productivity increased by > 30% because of the anti-apoptotic effect and increased specific productivity. These results demonstrate the potential use of this fusion protein as a supplement in mammalian cell culture during production of biopharmaceutical proteins.