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

Abstract

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.

Dynamics of unfolded protein response in recombinant CHO cells

“…Induction of GADD34 in HP can potentially reverse translational attenuation leading to increase in product titres and productivity. Corroborating this hypothesis is a recent study (Ohya et al 2008), where overexpression of ATF4 and GADD34 led to increase in productivity of thrombopoietin. Similarly, increase in XBP1 S levels indicates activation of Ire1a pathway.…”

“…This compares well with other studies on cell engineering of various chaperones and UPR pathway genes. For example, over-expression of GADD34, specific productivity of AT-III increased from 15 to 30 pg/cell/day (Ohya et al 2008). Similarly, over-expression of PDI in CHO cells increased productivity to about 22 pg/cell/day (Borth et al 2005).…”

“…The fraction of XBP1 S to XBP1 T mRNA increases slightly from day 3 to day 4, indicating further activation of the pathway. Presence of XBP1s has been observed in other recombinant CHO cell lines as well (Tigges and Fussenegger 2006;Ohya et al 2008). The presence of high levels of XBP1 S in an unstressed cell is a possible reason for the ineffectiveness of XBP1 overexpression on productivity (Ku et al 2008).…”

Engineering of chaperone systems and of the unfolded protein response

“…Expression of an humanized anti-CD154 antibody in CHO DG44 cells was only modestly enhanced by individual or combined overexpression of the cytosolic ATF6 fragment, XBP-1, or a fragment of eIF2a carrying the S51A point mutation (Ailor and Reff 2005). Expression of human antithrombin III or granulocyte-macrophage colonystimulating factor in CHO DXB11 cells was not enhanced by spliced XBP-1 (Ohya et al 2007). XBP-1 was spliced in antithrombin III expressing CHO DXB11 cells even before introduction of spliced XBP-1 cDNA (Ohya et al 2007).…”

Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

“…Indeed, yeast cells producing a GPCR were found to exhibit the hallmarks of a constitutively-active UPR [36]. In mammalian cells, activation of UPR-like responses has been shown to significantly improve recombinant protein production [37][38][39]. Similarly, in yeast overexpression of the genes encoding the Kar2 and PDI chaperones both reduced the UPR and increased heterologous protein production [40].…”