Construction of a fluorescent uricase fusion protein for characterization of urate transport
| Project/Area Number |
24790051
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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 |
Physical pharmacy
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| Research Institution | Tokyo University of Pharmacy and Life Science |
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Principal Investigator |
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| Project Period (FY) |
2012-04-01 – 2015-03-31
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| Project Status |
Completed (Fiscal Year 2014)
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| Budget Amount *help |
¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
Fiscal Year 2014: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2013: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2012: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
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| Keywords | 尿酸 / 尿酸トランスポーター / 蛍光タンパク質 / 腎臓 / 高尿酸血症 / トランスポーター / ウリカーゼ / 蛍光 / 蛍光プローブ |
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| Outline of Final Research Achievements |
Serum urate level is strictly regulated by renal urate reabsorption and secretion through the urate transporters. In this study, it was aimed to develop a high-throughput biosensing method for analysis of urate transporter. A fusion protein consisted of uricase and hydrogen peroxide-dependent fluorescent protein (HyPer) was constructed. Since uricase produces hydrogen peroxide when oxidizes urate, the fusion protein could detect real-time urate uptake with fluorescence.
Monkey renal epithelial-like Cos7 cells were co-transfected with fusion protein and urate transporter 1 (URAT1), and incubated 48 hours. The fusion protein/URAT1 co-transfected cells showed the significant increase of fluorescence when 15-200 micro M of urate was applied. The fluorescence was suppressed with 25 micro M of benzbromarone, that was a URAT1 inhibitor.
In conclusion, the constructed uricase-HyPer fusion protein could detect urate uptake specifically.
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Report
(4 results)
Research Products
(49 results)
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[Journal Article] Hydrophilic-interaction liquid chromatography–tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine–guanine phosphoribosyltransferase deficiency2014
Author(s)
Hiroshi Hasegawa, Yoshihiko Shinohara, Sayako Nozaki, Makiko Nakamura, Koei Oh, Osamu Namiki, Kiyotaka Suzuki, Akihiko Nakahara, Mari Miyazawa, Ken Ishikawa, Takahiro Himeno, Sayaka Yoshida, Takanori Ueda, Yasukazu Yamada, Kimiyoshi Ichida
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Journal Title
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
Volume: 976-977
Pages: 55-60
DOI
Related Report
Peer Reviewed
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