Hi all,
I have fused two proteins by overlapping PCR and have inserted a linker in between. I would like to insert also a NLS at the 3´ end. Is one NLS enough for transporting the proteins into the nucleus or do I have to add other multiple NLSs at different positions in the coding sequence?
Hi there,
If one refer to the first NLS described, PKKKRKV sequence of SV40 Large Antigen, one can say one NLS is enough (mutants in this sequence were no longer nuclear) but the motif has to be accessible for interaction with importin alpha.
I hope it will help.
Thanks a lot. So I think that adding the NLS to the 3´ would make it accessible to the importin. Are there any other portions of the protein where insertion of a NLS is unlikely to affect the protein activity but might be useful for increasing transport ? flanking the linker or at the 5`´?
Hi again,
If you have the 3D structure of your protein then you could consider the insertion of the NLS within a solvent-exposed loop (not involved in the biological function of the protein)... You could also have a go on the Nterminus of the protein instead of the Cterminus.
Hi Aimone,
We work on the application of site-specific recombination systems in plants to remove the unwanted selectable marker genes after genetic transformation. We add 1 NLS at the 3' end of the recombinase gene, so the protein (recombinase) can be efficiently targeted to the nucleus for working on the chromosomes. One NLS seems to work efficiently enough.
However, just for your information, some people place the NLS at 3' end, but others do choose to place NLS at the 5' end. The efficiency may be different depend on different genes (?). For this reason, for some of our projects, we would create a set (3) of constructs: one with one NLS at 5' end, one with one NLS at 3' end and another one with NLS at both 5'- and 3' ends (2 NLSs in total) for efficiency comparison. Good luck on your research.
Thank you very much Yuan for your comprehensive explanation! I will definitely follow your suggestion of inserting a NLS at both ends.
Aimone
Hi Alimone,
Recently, I came across this article about where to 'tag a peptide'--N-end, C-end or in the middle. Although it is not directly related to NLS tagging (for your question), but it does mention the role of NLS in the tagging of a peptide. It is kind of interesting. Thought maybe useful for you. See the attached article.
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