I have a blue copper protein, which I suspect may be binding an external chemical ligand in vivo. I am concerned that I may be losing this chemical ligand during purification. I normally purify this protein from the periplasm of E. coli via osmotic shock/lysozyme followed by dialysis followed by ion exchange column.
Hi there, I think the first question to ask is - What do you think the affinity of binding for this ligand is?
If you think the affinity is low (based on whatever ligand binding motifs you can identify in the gene sequence/structural data) then the interaction may be too transient and it may not be possible to co-purify with the ligand attached. Maybe you can simply add the ligand afterwards, if its identity is known.
If this ligand exists, you may be loosing this ligand at either dialysis or ion exchange. Maybe you should make an expresison construct with a 6xHis-tag on a terminus of the protein, then you might be able to get the protein pure enough using nickel affinity chromatography and perhaps will not need dialysis.
If that doesn't work, maybe you need to plan other experiments to show the existence of this interaction. Perhaps you could co-purify the ligand using said His-tagged protein, washing with cell lysate and sending anything additionally purified (exlcude protein and any non-specifically bound proteins) for mass spec analysis.
Before doing all of this I think you should definitely make sure sequence comparisons and previous literature suggest the existance of this interaction. If you still have an active protein, then maybe it is not necessary to do this and you should just work on with characterization
Hi Kiel,
Thanks for the suggestions. I thought of almost the same idea: using a His-tag to purify on a nickel column then running it through a desalting column and send the proteinless fraction for mass spec.
Regards,
Brian
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