I think you can add a NLS to the N- or C-terminus of your protein of interst if the protein does not have one. You may run the risk of interrupting a functional activity of your protein if you insert a heterologous sequence internally into your protein. NLS is just a short sequence of basic amino acid residuals.
Nuclear localization signals are generally small and pretty modular, so they can be added to protein sequences in a variety of ways (such as the N- or C-terminus). There also exist a wide range of classical NLS sequences, however, and these can have variable interactions with importin proteins in cells or function differently depending on their location within protein structures....so choose carefully which sequence to use and where to place it. Another wrinkle is whether you want a monopartite (such as T-Antigen's NLS) or bipartite sequence (such as nucleoplasmin's), which can affect the length of sequence or how you plan to clone things.
You can add a linker of small residues (like S and G) if you feel like the added flexibility is necessary. This may or but may not be important for protein-protein interactions or stability of your recombinant protein, you'd have to consider how the NLS you're using functions within its native protein. Empirically, 6-12 residues seems fine but this is something you should test during construction (will make any future cloning easier to troubleshoot).@Ying Chen
Can anybody share a paper describing NLS-attaching to the protein either N-terminal or C-terminal ? Will adding NLS to a protein completely push that particular protein to the nucleus ? Will that protein express itself only in nucleus ?
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