Andrew, over the years I have seen many papers where the approach you want to use has succeeded. As usual, the Flag tag, being extremely hydrophilic, is a great inter-protein linking peptide. If you already have it successfully attached to the peroxidase, I would leave that part as-is. If you are only using western detection, a direct connection to the 5HT6 will work with anti-Flag mAb M2 (M1 won't work). To increase the ability to bind a mAb in solution, it might be wise to open the gap between the two proteins with a glycine or two before the Flag, as Otmar H suggested. However, be careful about adding too many glycines. Polyglycine forms infinite beta pleated sheets with other copies of itself and is then highly insoluble. One or two glycines would probably be a good starting point.

If denatured (e.g. Western Blot), of course. Native or partially native techniques (protein arrays, luminex beads, indirect ELISA) should be tested on a case by case basis, but probably yes as single modules will fold on theirselves making your epitope tags sterically unhinded for your antibody.

It often depends on the protein. In our hands we have done transposon mediated insertions of GFP, FLAG and strep, so tags can integrate anywhere, and successfully pulled out protein complexes in native conditions. see http://www.ncbi.nlm.nih.gov/pubmed/21447707

good luck!

We have used a Preprotrypsin-FLAG-gene of interest construct (pFLAG-CMV3 from Sigma) which worked in our hands. Sometimes it may be helpful to add a small linker of 3-5 glycine residues to make the tag more flexible but there is no general rule. You have to test it for your specific protein and application.

It depends on the structure of the protein. You should design the postion of the tag on the surface of your enzyme to get accessible for affinity purification. Not too long tag is preferred to avoid the functional interference with your target protein

best regards

Gabor

If possible, you will have a starting point of an atomic model of the protein, and add some relatively small length of flexible linker residues. However, can you please clarify what you mean exactly by "multi-module fusions protein?" Do you mean an asymmetric protein complex, a multi-domain assembly, etc? I think that knowledge of the oligomerization state, other post-translational modifications and how surface accessible the insertion region of interest is are key. In many cases, loops between domains are not a problem. Can you tell us more about what kinds of proteins you're looking to tag? Are you speaking more generally or is this one particular case you're trying to troubleshoot? Making a small amount of protein for in vitro assays might also be useful.

Hi Andrew,

in general many tags can be used for your purpose. However, I would say that this depends more on your fusionprotein setup and you may have to try a few different configurations. We have used the tag54 described in the paper below succesfully as a linker for an scFv.

Rasche S, Martin A, Holzem A, Fischer R, Schinkel H, Schillberg S. One-Step Protein Purification: Use of a Novel Epitope Tag for Highly Efficient Detection and Purification of Recombinant Proteins. The Open Biotechnology Journal. 2011.

http://www.benthamscience.com/open/tobiotj/articles/V005/1TOBIOTJ.pdf

@Ashley: I am a bit sceptical about atomic modeling, we have had some surprises with that.

Internal epitope tags are the only solution when either N- or C-terminal fusions interfere with protein function or stability. As others have indicated, I would try and position the tag on a surface-accessible loop within a domain.

Ashley Pratt, I what I hope to do is clone a flag tagged peroxidase onto the C-terminus of the ciliary targeting sequence of the 5HT6 receptor protein, so that the final product will be N-5HT6-Flag tag-Peroxidase.

We had quite a positive experience with the myc tag, intercalating it within the subdomains of a complex surface associated molecule (see the attached pdf). Of course, if you know the structural details (i.e. domain organization) of your system this helps greatly. If you insert the tag in an inter-domain loop this makes things much easier. However, i totally agree with the statement that every tag should be tested depending on the specific system you want to analyze, as suggested by others before.

Article Insertion of a myc-tag within α-dystroglycan domains improve...

Hi Andrew - I think the most important question then is - are you trying to position the epitope tag so that it is intracellular vs. extracellullar - or is the tag just a means to an end for some other purpose? (I don't know if you're doing Westerns, microscopy, pulldowns, etc.)...There are so many structures of GPCRs these days, and I've done a lot of structural biology, which is why I suggest a homology model (especially to assess the accessibility of the flag tag based on it between squeezed between the receptor and peroxidase. Also important factors include - the receptor stoichiometry (is that known? best case of monomer will be the simplest clearly) and transgene expression level (will you control that to ensure all receptors have a given number of chimeric subunits - or is this a knockout system?) Do you already have a functional assay to ensure the downstream cytosolic functions you trying to probe/modify are intact still with such a large new module (i.e. interference with the G-protein coupled functions - sorry, I just don't know this system). I ask because whether you can "permit" the tag to be elsewhere is kind of a key concern. In solution structural biology, it's super common to prepare recombinant samples in vitro - which have internal tags - in order to find the right "happy" construct (as assayed by biochemical/biophysical means). But all this said, if you are just simply trying to tag your protein (i.e. you're set on this construct) for staining/fancy stuff, I'd mostly focus on varying the linkers between the receptor and the FLAG tag and the linkers between the FLAG and peroxidase (don't know its structure offhand). N, G and S are commonly good linker residues (though that may be debatable, depending on the protein and who you talk to. :)