I have cloned GFP at C-terminal of my protein to confirm its expression by GFP fusion
After end of my protein 2 aminoacids are present before start of GFP
I wanted to know whether this 2 a.a will interfere the expression of GFP.
Dear Elamurugan,
2 aa should not interfere with expression. However it is possible that fusion of your protein with GFP inhibits the GFP signal. This can be due to miscorrect folding of GFP when fused with your protein, followed, in same cases, by degradation of the whole fusion protein. To confirm your expression you can:
- put only GFP gene under the promoter of your gene instead of the fusion protein gene. This will confirm if expression occurs by your promoter, but not if your protein is stable
- make a fusion of your protein with a small tag (HA or His) and perform a Western. Being HA and His tag smaller than GFP, it is less likely (but non impossible) that tag influences the levels of your protein.
Enrico
I agree with Enrico, but you should also try to put the EGFP at the N-terminus of your protein of interest. It may well be that C-terminal EGFP leads to misfolding of your protein of interest and rapid degradation so that you will not be able to see teh EGFP fluorescence...
By the way, even a small epitope tag such as HA or His may interfere with folding and function of your protein of interest. Therefore, if C-terminal tagging of your protein of interest was not described so far in the literature you will have to generate N- and C-terminally tagged versions and compare expression patterns or use a functional assay for validation of protein function...
We always perform Western blots of our fusion proteins to confirm we're getting expression of protein the right size. An anti-GFP blot will also confirm that you've subcloned GFP correctly.
I agree with Jochen C Meier that the C terminal fusion could not only misfold GFP but also with the activity of your protein so its always better to have both N as well as C terminal Tags and see the consequences.....
I would follow Eric's suggestion. Answer the question of whether your fusion still results in production of GFP by performing a western with anti GFP ab.
We have successfully added GFP to the C-terminus of our receptor of interest and it functioned successfully. If your fusion is correct, the two amino acids should not have any negative impact unless you have generated a protease cleavage site which results in the loss of the GFP from the fusion when expressed in vivo.
Good luck
Mol Biol (Mosk). 2004 May-Jun;38(3):477-82.
if those two amino acids, don't disturb the "frame", then it will be ok, or else the protein product may differ largely.
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