2014 Fiscal Year Final Research Report
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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| Keywords | 尿酸 / 尿酸トランスポーター / 蛍光タンパク質 / 腎臓 |
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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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| Free Research Field |
タンパク質工学
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