Size and composition of spacer between protein and tag? - (Jan/05/2012 )

Hi,



I'm starting to classically clone my first construct. I need to append a tag, in this case 3xHA at the end of my protein. But surprise, surprise, even though this is a molecular lab, no one could advice me on size or composition of the spacer between my tag and the protein.

Any comments on that? Are there any rules of thumb? I haven't been able to find anything in the literature so far.

There are no good rules. Sometimes adding anything to the C terminus (or N terminus) inactivates the protein or makes it not fold correctly. You can sometimes look at the 3D structure to guess where insertions are possible (assuming you have a 3D structure). Blast can tell you if other homologous enzymes have extensions. Good linkers often include glycine/serine runs, often GGGGSGGGGSGGGGS or something similar to that. This provides flexibility from the glycines and moderate water solubility from the serines. The small size of the side groups reduces steric issues.

Thank you for the reply. I will of course check litterature to see if there are any trouble with my protein of interest, when tagging it.

Is there a reason that you don't mention Alanine as a good linker? Also, you mention a 15AA spacer, is it "recommended" to be that large? I had initially thought about 3-5AAs.

Yes, alanine is a good benign AA for linking. Often, no linker at all is used.

Hmm... I think I'll use your advice and look at the 3D structure and base my decision on that. Thank you very much!