Accurate detection of adenylation domain functions in nonribosomal peptide synthetases by an enzyme-linked immunosorbent assay system using active site-directed probes for adenylation domains
Project/Area Number |
26750370
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Research Category |
Grant-in-Aid for Young Scientists (B)
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Allocation Type | Multi-year Fund |
Research Field |
Chemical biology
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Research Institution | Kyoto University |
Principal Investigator |
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Project Period (FY) |
2014-04-01 – 2016-03-31
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Project Status |
Completed (Fiscal Year 2015)
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Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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Keywords | 非リボソーム性ペプチド / 非リボソーム性ペプチド合成酵素 / アデニレーションドメイン / 化学プローブ / ELISA / 酵素機能改変 / 合成分子プローブ |
Outline of Final Research Achievements |
A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we developed an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support.When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes.
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Report
(3 results)
Research Products
(31 results)
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[Presentation] Chemical Biology for modulators targeting cancer microenvironment and cell membrane signaling.2015
Author(s)
Kakeya, H., Yoshimura, A., Sugiyama, R., Kishimoto, S., Nishimura, S., Hattori, A., Ishikawa, F., Yomoda, Y., Lu, S., Katagiri, N., Shimada, S., Seto, E., Hayashi, H., Takahashi, N., Harada, H.
Organizer
Natural Products 2015; Natural product discovery & development in the post genomic era
Place of Presentation
San Diego
Year and Date
2015-01-11
Related Report
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