Is there some way to restrict a nuclear protein from going to the nucleus. I do not want to mutate/delete the NLS, rather would prefer to tag my protein to some cytoplasmic retention tag. Could you please suggest me any such tag or some other method to achieve the same.
I think the better choice is to use NES sequences, which basically are the same as NLS with the opposite outcome. There are some plasmids which carry, for example, GFP-NES sequences to tag proteins to the N-terminal. However, keep in mind that if you don't remove the endogenous NLS the localization of the protein could be affected
I think the better choice is to use NES sequences, which basically are the same as NLS with the opposite outcome. There are some plasmids which carry, for example, GFP-NES sequences to tag proteins to the N-terminal. However, keep in mind that if you don't remove the endogenous NLS the localization of the protein could be affected
yes indeed, the best it's to mutagenize the NLS and then add and NES
You can fuse your protein to the estrogen receptor ligand binding region. This is used in the CRE recombinase system, also designated as Cre-ER. The protein is then maintained in the cytoplasm until one adds tamoxifen, an estrogen analogue. Of course this assumes that the ER ligand binding domain will not affect the function of your protein.
I agree with Marie-Cecile, the best is to mutate your NLS. I would just add that if your protein is more than 50-60 kDa, mutating the NLS should be enough since your protein should not be able to passively diffuse across the nuclear pore complex.
After, you could just try to fuse a NES (like the one of the REV protein from HIV which is pretty strong). However, depending on the accessibility of the NES or NLS (to the import/export machinery), it might be efficient or not. There are no strict rules; it is more a matter of luck. But even if it seems to work (most of your protein is visible in the cyto), you might still keep a partial shuttling between the 2 compartments. So, be careful with the downstream analysis.
Today I leave from the house and I shall be away from February, 20th till March, 26th, 2015
thank you all for your inputs.. meanwhile I happened to find something called KDEL. Proteins that are supposed to stay in the ER lumen are marked by having an amino acid sequence called a KDEL sequence at their C-termini. This is a sign to the cell that these proteins are intended to stay in the ER, not get transported to other places. These are few research articles based on KDEL-
1). Cell, Vol. 48, 899-907, March 13, 1987. 2) Molecular Biology of the Cell
Vol. 14, 4114–4125, October 2003. 3) FEBS Letters 583 (2009) 3863–3871...
How do you think, this should work in our question?
http://oregonstate.edu/instruction/bi314/summer09/trafficking.html
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