| Project/Area Number |
15590380
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Tohoku University |
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Principal Investigator |
TOMITA Toshio Tohoku University, Graduate School of Agricultural Science, Associate Professor, 大学院・農学研究科, 助教授 (00126129)
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| Co-Investigator(Kenkyū-buntansha) |
KAMIO Yoshiyuki Tohoku University, Graduate School of Agricultural Science, Professor, 大学院・農学研究科, 教授 (00109175)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Staphylococcus aureus / gamma-hemolysin / two-component haemolysin / transmembrane pore / molecular architecture / 3-dimensional structure / electron microscopy / subunit arrangement / 二成分性溶血毒素 |
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| Research Abstract |
Staphylococcal γ-hemolysin (Hlg) is a two-component cytolysin secreated by Staphylococcus aureus. We have previously shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of approximately 200 kDa on human erythrocytes. Our recent study involving electron microscopy and chemical cross-linking suggested that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. In this research project, we attempted to construct nanogold-labeled glutathione-S-transferase (GST) fusion proteins of LukF and Hlg2 to visualize the alternate arrangements of LukF and Hlg2 under electron microscope. For nanogold labeling of the GST fusion proteins, three Cys residues (except for Cys138) of GST were successfully replaced with Ala by using primer extension procedure of polymerase chain reactions. Recombinant GST fusion proteins of LukF and Hlg2 thus obtained were hemolytically active in the presence of their counterpart. We now attempt to separate nanogold-labeled GST fusion proteins from unlabeled fusion proteins and nanogold particles. In this research project, we also analyzed fine structure of the heteroheptamers of Hlg by electron microscopy. The pore complexes isolated from human erythrocytes were negatively stained with phosphotungstic acid and observed under a transmission electron microscopy. High resolution images of the pore complexes from different angles were collected and analyzed. As a result, the pore complex of Hlg has a barrel morphology with 10-nm width and 10-nm height.
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