I am planning to tag a protein with mCherry using CRISPR/CAS9 method. I need to know if adding the mCherry seq. on 3'UTR can affect the functionality of protein or if I should add the mCherry before the gene.
I assume I need to check protein-protein interaction (or I may be wrong).
Are there any free tools to find any possible affect of the fluorescent protein on the target protein?
Like any other domain fused to a protein, the effects of fusing a fluorescent protein to either end of your protein depends on whether the termini of your protein play any functional and regulatory role. If fusions of other domains work, mCherry fusions should do so too. To be on the save side, I would try both orientations.
1) You can also try tagging with a very small 11aa part of split sfGFP or sfCherry (which is least likely to impair functions):
https://www.nature.com/articles/ncomms11046
2) A similar but bioluminescent tag (HiBiT) from Promega also may be interesting:
https://forums.promega.com/NanoBiT/thread/high-affinity-nanobit-we-are-looking-for-evaluators/
Thank you Annemarie.
Can you elaborate on how to find the functional roles of the termini? Can you recommend a tool? I am working with LGALS1 which is a small 15kd protein. I used Interpro tool and it shows one domain which is obviously functional. The mCherry is a larger protein and I don't know how to figure out if it will affect the protein function. (Interpro results attached).
Thank you Michael. The Split sfcherry looks very interesting. I am going to look into it!
Bioinformatics can only get you that far - As long as you have no 3D structure that tells you how close the termini are to potential interaction sites it remains guesswork. The residue annotations indicates a dimerization interface only a few residues from the C-terminus, which may or may not be perturbed by the presence of a fused domain. I still would try both orientations - it's unlikely that both perturb interactions in the same way (unless the two termini are right next to each other in the 3D structure), and if one of the two has an effect, it might actually make an interesting control.
I agree with Annemaria, In our lab we create similar constructs and we got sucess most of the time by adding (GGGS)2/3 linker between the protein and the tag.
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