Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression

To improve the production of recombinant human antithrombin III (AT‐III) in Chinese hamster ovary (CHO) cells, the genes encoding transcription factors, ATF4 (activating transcription factor 4) and XBP‐1s (the spliced form of X‐box binding protein 1), which were involved in the mammalian unfolded protein response (UPR), were cloned from CHO‐K1 cells. Overexpression of ATF4 significantly enhanced the production of recombinant AT‐III in CHO 13D‐35D cells. The specific rate of AT‐III production in the ATF4‐overexpressed CHO 13D‐35D cells reached approximately 23 pg/cell/day. After 144 h of incubation, the AT‐III concentration in the culture supernatant was twofold greater compared to that observed with parental CHO 13D‐35D cells. In contrast, ectopic expression of XBP‐1s failed to enhance the production of recombinant AT‐III in CHO 13D‐35D cells. RT‐PCR analysis revealed that high levels of XBP‐1s mRNA were present in the CHO cells, regardless of ectopic expression of XBP‐1s. Our results indicate that overexpression of the UPR transcription factor ATF4 is a promising means for improving the production of secreted protein pharmaceuticals in CHO cells. Biotechnol. Bioeng. 2008;100: 317–324. © 2007 Wiley Periodicals, Inc.