| Project/Area Number |
16K14712
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Project Status |
Completed (Fiscal Year 2017)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | イメージング / タンパク質 / 生物無機化学 / バイオイメージング / 蛍光エネルギー移動 / 低酸素応答 / 酸素 / ヘム |
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| Outline of Final Research Achievements |
We have developed genetically encoded FRET sensor for oxygen tension in the cell. Sea lamprey hemoglobin (slHb) is primarily monomeric in oxy form, while slHb self-associates to dimers upon deoxygenation. The oxygen dependent monomer-dimer transition of slHb was used for the FRET sensor for the oxygen. For the detection of the oxygen concentration, the single cysteine residues in the slHb were labeled by Alexa Fluor 488 or 594. Purified slHb with Alexa Fluor dyes were mixed and measured the fluorescence spectra. There was no FRET in our system when the oxygen pressure was 0.2 atm, while slHb with Alexa Fluor dyes exhibited FRET signal in a deoxygenated condition. This result suggested that slHb system is suitable for the genetically encoded oxygen sensor. Next, we examined a FRET measurement for slHb with fluorescent protein. However, the expression of the fusion protein was difficult in E. coli. Further optimization will be necessary for the genetically encoded FRET sensor protein.
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