| Summary: | The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an <i>Escherichia coli</i> expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP<sup>+</sup> ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.
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