| Co-Investigator(Kenkyū-buntansha) |
TANAKA Shigenori Seikagaku Corp., Dept. Functional Chemicals., Chief of LAL technology, 機能化学品事業部, LAL技術担当部長
KAWABATA Shun-ichiro Kyushu Univ., Dept. Biol., Associate Professor, 大学院・理学研究科, 助教授 (90183037)
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| Research Abstract |
In order to develop new detection methods for β-glucan, with high sensitivity, that is independent of the protease activity, we identified a β-glucan binding site in factor G. We expressed three kinds of domains in subunit α, in bacteria, as recombinant proteins. When β-glucan binding activity of the domains was examined, only the xylanase Z-like domain, which is localized in the COOH-terminal end of the subunit, exhibited the activity. We also examined inhibitory activity of the three domains on the activation of factor G by β-glucan. Again, only the xylanase Z-like domain showed inhibitory activity in a dose-dependent manner. Therefore, this domain was proved to have binding activity to β-glucan, thus functioning as a competitor for factor G activity through its β-glucan-binding. We furthermore measured binding parameters of the domain and β-glucan by using a BIAcore equipment. The xylanase Z-like domain showed a high Ka value of 8.03×10ィイD18ィエD1 MィイD1-1ィエD1 against β-glucans.
Since the xylanase Z-like domain is composed of two repeating units, each unit was separately expressed and measured their binding parameters. Each repeating units alone retained the binding activity, but their binding constant (Ka) was much lower.
In the present study, we identified a small protein fragment that has the β-glucan binding activity and simultaneously established the method to express the fragment as are recombinant protein in bacteria in a large quantity. We are currently developing new detection methods for fungus infection by detecting direct interaction for the fragment and β-glucan by a physicochemical method.
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