Has anyone had a problem with an Over Expression (O.E) line with either GFP, N or C terminal His or Myc tags. Did they affect the phenotype and if so, to what extent?
Daniel- first rule of toxicology, at the appropriate dose, all drugs will exhibit toxicity. Same applies for protein over-expression. Toxicity is more likely to be see due to the specific protein rather than the tag per se. However, toxicity of GFP-based tags, specifically induced photo-toxicity, is a well-documented phenomenon. I don't think a small epitope tag can be intrinsically toxic, but the protein to which it is attached might be.
In short, yes, I've seen toxicity with GFP-tagged proteins. I've also seen toxicity with untagged overexpressed proteins. Whenever possible, try to express your protein of interest an endogenous levels. If you are concerned about detecting function, knockdown the endogenous protein first, or use a knock-out or null cell line, when available. Overexpression studies are can be quite difficult to interpret, especially if you lack corroborating data showing a similar function using the endogenous protein.
So Daniel, to what extent will you trust an O.E results especially if one cannot not knockout the gene and then again, knockdowns also have their problems. How about looking at O.E in relation to qRT-PCR as a basis of gene function and thus phenotypic expression. Obviously you will like to attribute a reason to the differences you observe after the O.E and since that happens to be the only changes made to the organism, that should be the most plausible answer.
A lot depends on the nature of the protein being studied. Is it part of a multimolecular complex? If so, overexpression can disrupt these complexes and lead to inhibition of functions that the protein would normally promote. Alternatively, overexpression can lead to a protein bypassing normal mechanisms of regulating enzymatic activity or lead to supraphysiological levels of signaling/activity that may produce aberrant phenotypes. So in short, it is hard to predict whether overexpression phenotypes have a relationship to the normal role of a particular protein.
What kind of protein are you studying?
These are putative proteins with unknown functions. And yes we are trying to undertake both knockouts, knockdowns as well as O.E for complete functional analysis. However, we will be taking them one step at a time.
For putative proteins, you should try two tags in parallel, since epitope tags work best for immunoprecipation and Western blots, and GFP-tags are best for live cell imaging. If you can demonstrate the same overexpression phenotype using two different tags, that will be useful evidence that the observed phenotype is related to your cDNA, and not a tag-mediated artifact.
Protein localization in cells might be altered after tagging and over-expressing the protein, for e.g. nuclear protein may localize to cytoplasm after tagging. I have also seen differential post-translational modification of native and OE proteins. This may make your research difficult to interpret.
Kalpesh is right, but you may simply have to take this risk since you are focusing on predicted proteins. These overexpression experiments may be suggestive of subcellular compartmentalization or function, but will by no means will prove them.
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