2000 Fiscal Year Final Research Report Summary
The use of regulatory mechanism of glycolytic enzvme gene expression for production of recombinant proteins in animal cells
| Project/Area Number |
10559014
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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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 Kyoto Univ.Gaduate School of Biostudies, Professor, 生命科学研究科, 教授 (60077378)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAI Yoshito Kyushuu Univ.Faculty of Information Technology, Associate professor, 情報工学部, 助教授 (50175395)
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| Project Period (FY) |
1998 – 2000
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| Keywords | hypoxia / enhancer / lactate dehydrogenase / oxygen / gene expression / recombinant protein / animal cells / erythropoietin |
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| Research Abstract |
Oxygen supply is one of the major problems in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of human therapeutic recombinant proteins can be maintained or enhanced under low oxygen concentrations. A number of hypoxia-inducible genes have been found in animal cells and the induction in most cases is due to hypoxic activation of the gene transcription. A consensus sequence (HRE=hypoxia-response enhancer) responsible for the hypoxic activation exists in these genes and the binding of a protein, which is widely distributed in animal cells, to this sequence responding to hypoxia activates the promoter activity. The promoter of lactate dehydrogenase A gene is active in Chinese hamster ovary (CHO) cells and the vicinal HRE stimulates the promoter activity efficiently in hypoxia. We have prepared a number of permanent CHO cell lines producing recombinant human erythropoietin (Epo) under control of this promoter/HRE.Epo production was highly hypoxia-inducible when the wild-type of HRE was used but uninducible when the mutant HRE was used. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells cultured in 21% and 2% oxygen. Furthermore, forced expression of hypoxia-inducible factor-1α (HIF-1α) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even under low oxygen concentrations.
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