We are trying to generate a secreted red (or far-red) fluorescent protein construct in Drosophila. We had made some years ago such a construct with mRFP fused to a signal peptide, which worked OK, i.e. red fluorescence was detectable in the extracellular space, although signal was quite sensitive to bleaching. Now we tried to make a brighter and possibly more photostable version by fusing mKate2 as a fluor to the same signal peptide sequence that we had used successfully before. This construct does not appear to give any detectable fluorescence. We're not exactly sure what the problem is, but were suspecting that mKate2 might not fold or mature properly in the secretory apparatus, or that an oxidizing environment might affect the fluorophor. Does anybody have an opinion or experience on this? Any suggestion on which other red or far-rad fluorescent protein might be a better choice in this case? Thanks!
I don't know if this would be relevant advice, but in our model system (Arabidopsis) the sec marker is fused to DsRed and we get pretty bright stable fluorescence in the extracellular space. I don't know whether it would make a huge difference using DsRed, but i just wanted to give my 2 cents..
Thanks everyone, that is very helpful. Has anybody made an extracellular protein fusion with tagRFP-T?
Stefan, is it absolutely necessary that you use a secreted protein, or could something else potentially work given that it stains the extracellular space in red/far-red? Perhaps you could use a cyanine dye conjugated to an antibody?
Hi Carl, thanks for the suggestion. In this case we need to use a secreted protein.
mRFP1 and mCherry are being used to label endosoms, vacuoles and plasma membrane in plant model Arabidopsis thaliana. to my knowledge secretory system's pH and oxidations states are similar in both plants and animals.
ref: http://www.illuminatedcell.com/Vacuolarsystem.html
regarding TagRFP-T: this is several folds photostable compared to its predecessor FP TagRFP but lil less brighter. If you think your protein is very abundant in extracellular space go ahead with TagRFP-T otherwise TagRFP would be better choice.
the reason for low fluorescence of your mRFP construct can be due to low number of molecules reaching extracellular space (use brighter FPs like TagRFP or 2x /3x version of mRFP1) or stability issues of FP while moving through secretory path (low pH and oxidising environments) for later issue following article may give some direction to choose proper FP
http://www.nature.com/ncomms/2014/140602/ncomms4992/full/ncomms4992.html
all the best :)
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