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.
Ohya
Tomoshi
T
Advanced Medical Protein Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 2-25-1 Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan. ohya.tomoshi@md.mt-pharma.co.jp
Hayashi
Tetsuji
T
Kiyama
Eriko
E
Nishii
Hiroko
H
Miki
Hideo
H
Kobayashi
Kaoru
K
Honda
Kohsuke
K
Omasa
Takeshi
T
Ohtake
Hisao
H
D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1
C497109:ATF4 protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; D051701:Activating Transcription Factor 4; D000990:Antithrombin III