Protein protein interaction (PPI) of mTOR FRB domain and FKBP12 (FK506 binding protein) can be induced by Rapamycin. In this experiment, FKBP12 was fused to Ash-tag (FKBP12-Ash) and mTOR was fused to Monti-Red (mTOR-MR) or Azami-Green (mTORAG). Each color of mTOR construct was co-expressed with FKBP12-Ash then Rapamycin was added to the medium.
One of the outstanding features of Fluoppi is its reversibility. By adding PPI inhibitors, the pre-formed fluorescent Puncta would be dissociated in real time manner.This feature is perfectly suited for screening of PPI inhibitors in each phase of drug discovery program.
Using p53-Ash-tag/MDM2-Monti-Red, Nutlin-3, a p53/MDM2 PPI inhibitor, was added to the medium after confirming the formation of Puncta. Dissociation of Puncta were observed in 10 minutes after the addition of Nutlin-3.
mTOR (FRB domain) and FKBP12 is a well known PPI whose interaction is induced by Rapamycin. On the other hand, mTOR (FRB domain) and mCAB (the BBH region of Calcineurin A (residues 340-394) fused to Calcineurin B*) interaction is induced by FK506. In this experiment, The mCAB-hAG, mTOR-Monti-Red and Ash-FKBP12 were cotransfected into the cells at the same times. When the cells were exposed to Rapamycin, only the red Puncta were formed as shown in the left 4 pictures. On the other hand, in the case of FK506, only the green Puncta were detected. The results demonstrate the possibility that multi-color Fluoppi could be applied to visualize branched signaling pathway.
* Reference:P. A. Clemons et al. Chem. Biol. (2002)
Monti-Red is a new red fluorescent protein that is developed for Fluoppi technology.
Among the many red fluorescent proteins, Monti-Red is relatively bright, allows clear observation of Puncta under a fluorescence
microscope, and is ideal for use in long-term imaging.
Oligomerization
|
Excit./Emiss. Maxima (nm)
|
Molar Extinction
Coefficient (M-1cm-1) |
Fluorescence
Quantum Yield |
Brightness
|
pH sensitivity
|
Cytotoxicity
|
---|---|---|---|---|---|---|
Tetramer
|
571 / 607
|
83000
|
0.3
|
24.9
|
pKa=5.5
|
No
|
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