In almost every article researchers say, "we tagged protein x with GFP by using transiently transfected vector y" but they seldom explain the effect of expression levels of engineered protein and its biological effects.
If, let's say, I want to tag a protein whose intracellular level affects organelle morphology (for instance Mfn2 increases mitochondrial fusion) how can I achieve tagging the protein without flooding the biological system with excess amount of protein of interest (e.g. Mfn2). In other words, how can I tag protein of interest with GFP without overexpressing it?
transient transfection vectors are all containing strong promoters for high level expression of proteins of interest. This is good for studies involving protein isolation, but how can I tag a protein with GFP in the cells by transient transfection?
I hope it was clear enough to express my question.
Thanks for the answers in advance
Hi Atakan...
I also agree with your concerns about the biological effects of overexpressed proteins.......In my opinion ..Cloning and expression are affected by two aspects.....1) the copy number of the vector one is using ..and 2) the promoter used for expression (strong, inducible etc.)..... Hence the fine tuning of the expression may be achieved by either choosing a low copy or single copy expression vector.....and also by using a promoter which is not very strong ... One can also go for literature where people may have used promoters from the system being study.. whose levels are not vey high and they may be just suitable to achieve the level of expression similar to biological levels and just OK to track theGFP fused protein.........something tailormade to your requirements may or may not be available...and if not ....the idea is definitely worth trying..
Hi, Atakan, one (laborious) way to circumwent the problem of overexpression is of course the generation of a knock-in mouse... else you can run western blots to determine the levels of your fluorescencently tagged protein and a run against the protein itself (labeling the tagged and non-tagged copies) and estimate the expression ratio...
Like what Ajay said, put GFP in a weaker promoter if you're having difficulty. If you don't want to do that, I would recommend a time course. There is no reason that you need to overexpress for 16 hours or more. Sometimes, you can get away with as little as one hour with a strong GFP promoter in a perfect cell. If you don't want to change protocols, I would suggest transfecting, then taking a sample every hour for 8 hours and western blot for GFP. At some time t, the proteins should reach a steady-state level which will represent your maximum. Now the problem for this is it is a bulk measurement instead of a single cell analysis. It will still get you useful information, though, for how long to do your experiment.
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