The calculated Forster distance (R0= 5.25 nm) for the TagBFP-TagGFP2 pair is larger than those reported for the standard ECFP-EYFP and mCyPet-mYPet pairs (R0= 4.86 nm and 4.93 nm correspondingly).
Calculation of FRET efficiency, E, based on the increase of donor emission upon cleavage of the fusion protein in vitro produced E=0.57 for TagBFP-TagGFP2.
For comparison, for EBFP2-TagGFP2 pair E=0.38; for ECFP-EYFP pair E=0.42; and for mCyPet-mYPet pair E=0.51.
When TagBFP and TagGFP2 free proteins were coexpressed in HeLa cells, the cross-bleed-corrected FRET normalized to fluorescence of donor was 0.85%.
Under the same experimental conditions, the FRET between ECFP and EYFP free proteins coexpressed in HeLa cells was 6.2%, confirming their weak dimerizing tendency.
Thus, the TagBFP and TagGFP2 proteins derived from the different marine sources and, as a result, lacking ability to form heterodimers, provide more than 6-fold lower background for FRET analysis than the weakly dimerizing FRET pairs, such as the ECFP-EYFP.
Taking altogether, this data suggests that TagBFP-TagGFP2 pair is not only superior to other BFP-GFP pairs but is one of the best among available FRET pairs of the true monomeric fluorescent proteins [Subach et al., 2008].