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Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer

De, Abhijit ; Gambhir, Sanjiv Sam

The FASEB journal, 2005-12, Vol.19 (14), p.2017-2019 [Periódico revisado por pares]

United States: Federation of American Societies for Experimental Biology

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Título:
Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer
Autor: De, Abhijit ; Gambhir, Sanjiv Sam
Assuntos: Animals ; Energy Transfer ; green fluorescent protein ; Green Fluorescent Proteins - metabolism ; Luciferases, Renilla - metabolism ; Luminescent Measurements - methods ; Luminescent Proteins - chemistry ; Mice ; Microscopy, Video ; Nanostructures ; Photons ; Protein Binding ; Protein Interaction Mapping ; Protein Structure, Tertiary ; rapamycin and optical CCD imaging ; Recombinant Fusion Proteins - metabolism ; Renilla luciferase ; Sirolimus - pharmacology ; Tacrolimus Binding Protein 1A - metabolism
É parte de: The FASEB journal, 2005-12, Vol.19 (14), p.2017-2019
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
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Descrição: ABSTRACTThis study demonstrates a significant advancement of imaging of a distance‐dependent physical process, known as the bioluminescent resonance energy transfer (BRET2) signal in living subjects, by using a cooled charge‐coupled device (CCD) camera. A CCD camera‐based spectral imaging strategy enables simultaneous visualization and quantitation of BRET signal from live cells and cells implanted in living mice. We used the BRET2 system, which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC (DBC) as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. To accomplish this objective in this proof‐of‐principle study, the donor and acceptor proteins were fused to FKBP12 and FRB, respectively, which are known to interact only in the presence of the small molecule mediator rapamycin. Mammalian cells expressing these fusion constructs were imaged using a cooled‐CCD camera either directly from culture dishes or by implanting them into mice. By comparing the emission photon yields in the presence and absence of rapamycin, the specific BRET signal was determined. The CCD imaging approach of BRET signal is particularly appealing due to its capacity to seamlessly bridge the gap between in vitro and in vivo studies. This work validates BRET as a powerful tool for interrogating and observing protein‐protein interactions directly at limited depths in living mice.
Editor: United States: Federation of American Societies for Experimental Biology
Idioma: Inglês

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Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer

De, Abhijit ; Gambhir, Sanjiv Sam

United States: Federation of American Societies for Experimental Biology

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