| Project/Area Number |
07559009
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
広領域
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SASAKI Ryuzo Graduate School of Agriculture, Kyoto University, Professor, 農学研究科, 教授 (60077378)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAI Yoshihito Faculty of Computer Science and Systems Engineering, Kyusyu Institute of Technol, 情報工学部, 助教授 (50175395)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥18,500,000 (Direct Cost: ¥18,500,000)
Fiscal Year 1997: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1996: ¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 1995: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | Recombinant protein / Erythropoietin / Hypoxia / Animal cells / Carbohydrate structure / Enhancer / Cell culture / Glycoprotein / 発現調節 / 組み換え型蛋白質 / 低酸素エンハンサー / プロモーター / レポーター / 生合成 / 組換え型エリスロポエチン |
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| 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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