I over-expressed HA tagged mammalian protein (27KD) in 3 different cell lines; however, I saw the over expressed protein (exogenous) behaves differently than the endogenous protein in the all cell lines that I used. For instance, the exogenous protein expression level depends on other protein expression or proteosome inhibitor. I checked DNA sequence of the protein and there were no problems. The exogenous protein can be detected by Western blotting using either anti-HA or anti-protein antibody.
Is there any ideas why the over expressed protein behaves differently than the endogenous protein? Any ideas or suggestions would be appreciated.
Ahmed
The protein may have difficulty folding properly when overexpressed because of a limited supply of suitable chaperones, or of a binding partner. If the protein requires post-translational modification, the enzymes required may not be present in sufficient quantity. The HA tag may interfere with proper folding, or the construct may lack the appropriate subcellular targeting signal.
I agree with Adam's answer above. I'm not a fan of using tags or fusion proteins as they can create problems like this. The tag doesn't just sit there doing nothing. It can actually cause problems with proper shuttling, binding partners, folding, etc. If you can go without an HA tag, I'd recommend it.
I do agree with above answers, most of the protein folding and post-translational modifications are most authentic in mammalian cells expression system and it works well for secreted and membrane proteins. However, the presence of tag cause the improper folding and also binding of chaperones that leads to unusual behavior of these over expressed proteins.
Thank you very much for all. I know post-translation plays a major role with protein functions, but why couldn't the over-expressed protein get it even if it was over-expressed in the same cell host and in different expression levels?Is the any way by which I can check the appropriate subcellular targeting signal? By the way, the overexpressed and endogenous proteins localized almost similarly in different cell compartments.
Thanks again
Ahmed
I agree with all the answers above. Might I add that when we used HA tags on some proteins (and it depends on the protein), the HA tag caused what we discovered to be aggregation of the protein when it was over expressed. A less rigorous promoter might help in the case of some proteins (kind of doubt it, but it did help in one instance), but HA tags can produce several sorts of problems in addition to the ones mentioned above. Your localization comment made me think of our past problems with aggregation. I would suppose it might cause aggregation with other proteins at very high levels of expression.
Best wishes for your work.
Ahmed, though the protein appears to be localized correctly, it may not be folded or have undergone correct post-translational modifications (phosphorylation, etc) that creates a functional protein. I'm not sure if I understand your question correctly. Are you upregulating the protein in a cell line that already endogenously expresses it, and if so to what extent does the cell line express it? Highly or just a little bit? To determine expression at various subcellular localizations, I would a nuclear lysate prep, a cytoplasmic lysate prep and a cell membrane lysate prep in order to determine where in the cell your protein is expressed. You should be able to find protocols to do these different lysate fractions online.
Thanks all! Jenny, Yes I overexpressed the protein in a cell line which already endogenously expressed the protein. The 3 cell lines that I used can expressed the protein in different levels from really low to really high (much higher the endogenous protein). I fraction cells into different parts such as, cytoplasmic, nucleus, chromatin binding protein, active and inactive chromatin binding protein and saw both proteins endo and exogenous distributed almost similarly in all theses fractions.
Ahmed
There are many limiting factors inside cell which can restrict the PTM of higher amount of proteins. Some of them are listed in all the above answers. Apart from these, any tag added to original protein (even if it is very small) may push it towards functional inactivity or diversity. The localization might not be affected much as it depends upon complete signal sequence of the protein. But functional characteristics depends of proper conformation of active site which depends upon folding and PTM of protein.
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