| Project/Area Number |
16310147
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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 | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | The University of Tokyo |
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Principal Investigator |
HAMAKUBO Takao The University of Tokyo, Research Center for Advanced Science and Technology, Professor, 先端科学技術研究センター, 教授 (90198797)
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| Co-Investigator(Kenkyū-buntansha) |
SAKIHAMA Toshiko The University of Tokyo, Research Center for Advanced Science and Technology, Specially Appoimted Associate Professor, 先端科学技術研究センター, 科学技術振興特任教員(特任助教授) (70187061)
MASUDA Kazuyuki The University of Tokyo, Research Center for Advanced Science and Technology, Specially Appoimted Research Associate, 先端科学技術研究センター, 科学技術振興特任教員(特任助手) (20345151)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥15,700,000 (Direct Cost: ¥15,700,000)
Fiscal Year 2005: ¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 2004: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Keywords | baculovirus / membrane protein / GPCR / γ-secretase / lipid raft / nicastrin / peptide transporter / FRET / 核内受容体 / 磁性ビーズ / プロテオミクス / 転写複合体 / HNF4α / ラフト / スタチン / アルツハイマー |
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| Research Abstract |
Membrane proteins such as peptide transporter G-protein coupled receptors (BLT1, CCR2, CCR5, Edg1, AT1a, AT2, D1, D5) and their complex (reoeptor-Gs-adenylyl cyclase) or active γ-secretase complex (presenillin-APH1-nicastrin-PEN2) have been successfully recovered in baculovirus fraction. The expressed membrane proteins were considered to be active on the baculovirus membrane. Interestingly, γ -secretase acitivity was recovered only when all 4 components were expressed on the baculovirus. Using these membrane protein expressed baculovirus, specific monoclonal antibodies (Mabs) were generated against membrane proteins including GPCR. These Mabs are usable not only for western blotting but also FACS analysis and immunohistochemistry. Some of them have shown the neutralizing activity against ligand-receptor mediated cAMP production or the antibody dependent cell cytotoxic activities. Also we showed the importance of lipid micro- environment of membrane and geranyl-geranylation for γ-secretase acitivity in generating β-amyloid from its precursor. One molecule FRET(fluorescence resonance energy transfer) was succefully constructed using RGS (regulator of G- protein signalling) and Gi which recognized G- protein activation by alminium fluoride.
These results indicate that the baculovirus display system of membrane proteins or their complex is useful not only for high throughput screening assay system but also for making active Mabs for therapeutic use.
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