|Protein (state)||t1/2 (s)||Power||Light||Mode||In Cell||Fusion||˚C|
Because Aequorea victoria green fluorescent protein (GFP) is relatively indifferent to N- or C-terminal fusions, we eventually generated mRFP1.3 by replacing the first seven amino acids of mRFP1.1 with the corresponding residues from enhanced GFP (MVSKGEE) followed by a spacer sequence NNMA (numbered 6a–d), and appending the last seven amino acids of GFP to the C terminus. mRFP1.3, unlike its predecessors, was found to have an equivalent high level of fluorescence regardless of fusions to its N terminus.
Through additional rounds of screening random libraries based on mRFP1.3 and wavelength-shifted mRFP variants, we identified the beneficial folding mutations V7I and M182K, which were incorporated into clone mRFP1.4.
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.