The protein has an N-terminal tag (single copy) and is being purified from insect cells (Sf9). We have been unable to elute it from FLAG affinity beads using either 1X FLAG peptide or pH3 glycine (estimate 0.5 mg bound to 1 mL bed volume). The protein is destined for structural studies so a mild elution is necessary (and retention in pH3 glycine is a concern - leading me to question potential efficacy of 3X FLAG peptide). Has anyone ever tried to elute captured protein via enterokinase treatment? I suspect steric occlusion will limit success - but happy to hear from anyone that has tried.
Hi there,
As you mentioned the success of the digest will be dependent on cleavage site accessibility... Which can't be anticipated if the structure of the protein is not known. In my experience, I've managed to efficiently recover protein (reluctant to elution with free competitor) from tag affinity column using thrombin cleavage.
Hi David,
I think that the enterokinase treatment could work (if the tag is well exposed). Here a reference in which this approach has already been used.
It could be helpful use a His-tagged enterokinase in order to further separate it from your protein of interest.
Thank you for the thoughtful responses. There is no crystal structure available for our protein, but we can make some reasonable assumptions as it belongs to the kinase superfamily. To clarify, there is a FLAG tag (DYKDDDDK) at the amino terminus, so the enterokinase cleavage site (DDDDK) is built into the tag and should liberate an untagged protein following treatment. Becasue we have shown that all of our protein is now "stuck" to the anti-FLAG antibody/resin, enterokinase would need to essentially "compete" with the antibody in order to recognize and cleave the protein. In afterthought, this is very unlikely and would (likely) minimally require us to engineer a spacer into the recombinant protein (as suggested above). I prefer not to take this route so we'll explore some other options, including alternative affinity resin provider(s).
Hi David,
Have you figured it out? I am also struggling with a flag-tagged protein complex that we need for structural studied, that will not come off the beads.
Hi Daniel,
This is certainly a rather dusty relic you found in the attic - I may have to print it and file it with my vinyl LP collection ;-)
To answer your question - no, we never did solve that challenge. But I suspect there were some unnecessary challenges we had subjected ourselves to. Most notably, we were trying to bind and elute in bulk. This wasn't for any particularly good reason, we simply didn't have access to proper chromatography equipment or supplies. I think such an approach creates a battle with on/off equilibria - if your protein is bound to beads in a column with a moving solvent front, I want to believe that you change these equilibria dynamics, though its outside of my knowledge base to propose why or how.
The other point to make is that we weren't paying attention to the composition of the beads. Now that we do have proper chromatography equipment, we understand that not all beads are created equal. Some proteins will stick non-specifically to sepharose-based beads but not to their nitrilotriacetic acid (NTA) counterpart. So if you do have an issue, try a different matrix and see if that solves the problem.
Hope that helps!
David
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