C. elegans TAP tags without GFP and His

Model Organism: Worms Methods & Reagents
1

Hi.

I am looking for tandem affinity purification (TAP) tags that have been used in C. elegans, and I found several including LAP tag, HA-Hisx6-Myc tag and so on.
But all the tags I found include GFP or His, which I cannot use in my experiment.

Does anyone know of TAP tags without GFP or His?

I appreciate your help in advance.

2

I know that the Boulton lab used the HA-8xHis-TEV-Myc tag in to purify CeBRC-1 and Ce-BRD-1 complexes:
http://www.ncbi.nlm.nih.gov/pubmed/15152596
http://www.ncbi.nlm.nih.gov/pubmed/16628214

Now I realize that you do not wish to use an 8xHis tag; this purification protocol relied on the HA and Myc tags. The first step was using Protein A coupled anti-HA antibody(MAb12CA5), followed by elution with 2xHA peptide. Then Protein A coupled anti-Myc antibody (MAb9E10) was used. The complex was cleaved off the beads with TEV protease. You could probably either generate a similar tag yourself and leave out the 8xHis tag, or get the vector (which I have) and use Gibson cloning to swap in a flexible linker in place of the 8xHis tag.

That said, I don’t really like the HA and Myc tags all that much. I prefer 2xFLAG tags (easier to compete off beads with peptide). FLAG-Myc could work, but leave a TEV cleavage site between the epitopes, since it is very difficult to compete Myc-tagged proteins off beads.

Another alternative that looks appealing (though I have not tried) is using Streptavidin one-step purification. You could add the 15-23 amino acid biotin acceptor peptide to your protein of interest (could add a 2xFLAG tag if you wanted a 2nd tag). The BirA biotin ligase would need to be co-expressed. This approach was used mammalian cells and transgenic mice for one-step complex purification (http://www.ncbi.nlm.nih.gov/pubmed/17093407; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164612/).

Additionally, Steve Henikoff has used this approach to purify cell-type specific nuclei using biotin-acceptor peptide tagged nuclear pore protein (NPP-9) with a 3xFLAG tag http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317158/. His his-72 driven BirA biotin ligase strain is available from the CGC (http://www.cgc.cbs.umn.edu/strain.php?id=17660). Of the above-mentioned approaches, I would suggest either one step streptavidin, or a sequential FLAG-Streptavidin purification.

best wishes,

Jordan

3

I think I can try FLAG-TEV-Myc and FLAG-TEV-streptavidin first. Thank you very much for your advice.

  • Baek