| Project/Area Number |
11555220
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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 |
生物・生体工学
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| Research Institution | KOBE UNIVERSITY |
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Principal Investigator |
FUKUDA Hideki Kobe University, Graduate School of Science and Technology, Professor, 自然科学研究科, 教授 (30263396)
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| Co-Investigator(Kenkyū-buntansha) |
TACHIBANA Yoshihisa Nagase & Co., Ltd., Research & Development Center, Researcher, 研究開発センター, 研究員
YAMAJI Hideki Kobe University, Faculty of Engineering, Research Associate, 工学部, 助手 (40283874)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥10,000,000 (Direct Cost: ¥10,000,000)
Fiscal Year 2000: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1999: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Keywords | insect cells / Sf9 / baculovirus / recombinant protein / immobilization / 組換えタンパク質生産 / 組換えタンパク質 / 細胞保持粒子 / ポリビニルホルマール樹脂 |
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| Research Abstract |
Sf9 insect cells infected with a recombinant baculovirus expressing β-galactosidase and suspended in fresh medium at the time of infection were cultured in shake flasks. The effects of cell density and multiplicity of infection (MOI) on β-galactosidase production were quantitatively analyzed by plotting the β-galactosidase yield against the time integral of the viable cell density from the time of infection to the time when the β-galactosidase production reached a plateau. The β-galactosidase yield had a maximum value at a comparable viable cell density time integral for each MOI tested. Since glucose and fructose were exhausted when the culture reached the cell density time integral, protein production in a high-cell-density culture was limited by nutrient depletion in the culture medium. In cultures infected at a low MOI (≦1), the specific productivity, and thereby the yield, of β-galactosidase declined with decreasing MOI.Therefore, recombinant protein production can be optimized if
… More
the cells are infected at a high MOI (≧1) and at a cell density such that the cell density time integral reaches the viable cell density time integral just as the protein production is completed.
Immobilization of Sf9 cells using porous biomass support particles (BSPs) and production of β-galactosidase by the immobilized cells after infection with the baculovirus were then investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal resin BSPs (2 mm cubes) with matrices of 60 μm mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5×10^7 cells/cm^3-BSP in cultures with regular replacement of the culture medium. After infection with the baculovirus, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5×10^7 cells/cm^3-BSP were infected with the baculovirus, efficient β-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion. Less
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