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.
Subject(s)
Activating Transcription Factor 4/metabolism , Antithrombin III/biosynthesis , CHO Cells/metabolism , DNA-Binding Proteins/metabolism , Genetic Enhancement/methods , Nuclear Proteins/metabolism , Protein Engineering/methods , Activating Transcription Factor 4/genetics , Animals , Antithrombin III/genetics , Cricetinae , Cricetulus , DNA-Binding Proteins/genetics , Humans , Nuclear Proteins/genetics , Recombinant Proteins/biosynthesis , Regulatory Factor X Transcription Factors , Transcription Factors , X-Box Binding Protein 1ABSTRACT
To improve the production of recombinant human antithrombin III (AT-III) in Chinese hamster ovary (CHO) cells, the gene encoding growth arrest and DNA damage inducible protein 34 (GADD34), which is a transcription factor involved in the unfolded protein response (UPR), was cloned from CHO-K1 cells. Overexpression of GADD34 significantly enhanced the production of recombinant AT-III in CHO 13D-35D cells. The specific rate of AT-III production in the GADD34-overexpressing CHO 13D-35D cells reached approximately 28 pg/cell/d. After 144 h of incubation, the AT-III concentration in the culture supernatant was approximately 40% higher than that observed in the case of the parental CHO 13D-35D cells. The mRNA expression, specific activity, and fucosylation of AT-III were not affected by GADD34 overexpression. Overexpression of GADD34 is a promising method of improving the production of secreted protein pharmaceuticals in CHO cells.
