|Ex λ||Em λ||EC (M-1 cm-1)||QY||Brightness||pKa||Maturation (min)||Lifetime (ns)|
|% Normal Cells||OSER/NE ratio||Cell Type||Reference|
|95.0 ± 0.8 (10000 cells)||-||HeLa||Cranfill et al. (2016)|
|91.0 (98 cells)||-||HeLa||Costantini et al. (2015)|
|80.0 (369 cells)||2.0 ± 0.3 (38 cells)||U-2 OS||Bindels et al. (2016)|
|69.0 (1130 cells)||-||U-2 OS||Manna et al. (2018)|
|t1/2 (s)||Power||Light||Mode||In Cell||Fusion||˚C||Reference|
|68.0||Shu et al. (2006)|
Randomization of position 163 in mRFP1.4 led to the identification of the substitution M163Q, which results in a nearly complete disappearance of the absorbance peak at ∼510 nm, present in all previous mRFP clones.
To test whether the introduction of GFP-type termini into mRFP variants would benefit fusion proteins expressed in mammalian cells, we fused mRFP1 and mCherry to the N terminus of α-tubulin. In most HeLa cells, expression of mRFP1-α-tubulin resulted in diffuse cytoplasmic fluorescence rather than proper incorporation into microtubules. However, mCherry-α-tubulin fusions were successfully incorporated into microtubules in most cells, similar to results seen with GFP-coupled tubulin.
This strongly suggests that the fluorescent protein tag caused clustering artifacts (Fig. 2a) and that ClpX and ClpP fluorescent protein fusions cannot be trusted for determining localization of native, untagged proteins. [...] These findings motivated us to evaluate other fluorescent proteins fused to ClpP or ClpX. We found that sfGFP, Venus, mCherry and mCherry2 all caused substantial foci formation, despite being monomers or very weak dimers when expressed alone. mKate2 and TagRFP-T caused intermediate clustering, whereas with mVenus and mYPet most of the fluorescence signal was spatially uniform, although we observed foci in a few cells. mTagBFP and mEos2 fusions resulted in a weak signal with infrequent dim foci. We detected no foci for photoswitchable (PS)-CFP2, reversible switchable (rs)FastLime (data not shown) and a mutant of sfGFP with a charge of −30 (GFP(−30)), but the signals were very dim. Finally, mGFPmut3, Dronpa and Dendra2 displayed an essentially uniform signal. Fluorescent protein fusions to ClpP generally caused more foci formation than fusions to ClpX, in particular for mYPet (Fig. 2d).