My experiment deals with adding a SV40-NLS to my protein of interest but it has already 3 Nuclear Exclusion Signals. I added one NLS signal to the N-terminal of my gene (with a comet-GFP tag), without removing the NESs. But am not able to improve the nuclear localisation [See pic attached of ].
I am thinking of either:
1.Adding one more NLS to the C-terminal, or,
2. Adding one more NLS before the N-terminal NLS
Any ideas or suggestions are helpful.
Adding another NLS might indeed help. It could also be worthwhile to add a GR and treat with dexamethasone to induce active shuttling to the nucleus.
Hi Jos. Thanks for the reply. Could you elaborate on your answer - as in, what is GR and whether I should add NLS to the other terminal i.e. C-terminal or before the N-terminal NLS. Thanks
What do you mean "part of a major sequence of my protein"? If adding more NLS does not help, perhaps you could use site-specific mutagenesis to substitute residues within the NES regions and thereby weaken or even eliminate an NES, two, or all three. Properly controlled, you may be able to determine if it affects whatever property of your "major sequence" that is of concern to you while eliminating the exclusion property.
I edited it Mike. It made my question confusing But thanks for your suggestion.
Thank you your answers.I am wondering what would be more helpful - adding a SV40 NLS or a bipartite NLS? [Just to recap, I want to improve nuclear localization of my protein that has 3 Nuclear exclusion signal sequences in it. I tried by adding one NLS to the N-terminal and observed for nuclear localisation but its just 40%. In order to improve ths, I am thinking of adding one more NLS]
On the GR: http://en.m.wikipedia.org/wiki/Glucocorticoid_receptor
I personally have no experience with bipartite NLSs. Though mutating the NESs in your sequence might just do the trick...
Dear Jos,
So I don't know that exact location of NES, just know the regions where they may be. To begin with,I found some lucine-rich regions and I am planning to mutate them first and see if they affect the nuclear localisation? Do you see this approach working? Plz suggest any other ideas that you may have.
That might work.. Keep in mind though that the folding properties and activity of the protein might change when you mutate the sequence.
So can i facilitate export of protein from nucleus by adding a conserved NES motif to my protein????
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