| Research Abstract |
Mammalian cells are used for producing recombinant proteins whose post-translational modifications are important for therapeutic efficacy. Erythropoietin (EPO), a glycoprotein, is a representative of these. Oxygen is a limiting nutrient in animal cell culture and its supply is still worthy of improvement for producing useful proteins with a high efficiency. In mammalian cells, a number of hypoxia-inducible genes have been found. Induction of these is mainly due to activation of transcription by binding of a transcriptional activator, a hypoxia-inducible factor-1 (HIF-1), to the hypoxia-response enhancer (HRE). In this project, we developed the animal cell culture by which recombinant proteins can be produced with a high efficiency under either normoxic or hypoxic conditions, using a variety of Promoters and HRE,and EPOcDNA as reporter. Using Chinese hamsterovary (CHO) cells, we show that, under hypoxic condition, the HRE can activate not only the promoter of EPO gene but also promoters of CMV and EF-1alpha genes, both of which are very active in animal cells. LDHA gene is also hypoxiainducible. We prepared CHO clones harboring the plasmid in which transcription of EPO cDNA is under control of HRE and the promoter of LDHA gene. We examined oxygen-dependent production of EPO by CHO cells and compares characteristics of EPO produced in various oxygen concentrations, proposing a new method for production of recombinant proteins by which one is allowed not to be obsessed with oxygen supply.
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