2015 Fiscal Year Final Research Report
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 |
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| Research Field |
Chemical biology
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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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 |
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| 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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| Free Research Field |
ケミカルバイオロジー
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