Hello everyone,
I am currently working on a protein that is 10x his-tagged and positively charged (predicted pI=10.16). But when I tried to use Ni-NTA column to purify the protein, it's not binding to the resin at all. Then I switched to cation-exchange chromatography, but this protein won't bind SP-sepharose resin either. Does any one have any suggestion?
Many thanks!
This problem is often the result of the protein being highly aggregated. It sometimes helps to reduce the rate of expression by lowering the culture temperature (for expression in bacteria) or co-expressing with chaperones.
You can find out if the protein is highly aggregated by running it through an appropriate size exclusion column and comparing the elution volume to the void volume.
Actually, it is not uncommon for an overexpressed protein to form soluble aggregates.
Without knowing the details of the CEX application, it could be hard to adsorb and elute the protein with a relatively high pI. Method optimization is critical to purify your POI when the protein is highly alkaline. Binding buffer and injection buffer composition and conditions (such as pH and salt) are considerably important in CEX.
For the IMAC approach, it may be related to histag binding conformation. Is the tag reachable by the Ni ligand? You may switch the position of the tag at the expression level to observe if any improvement. You pinpointed this is not a case of solubility (inclusion bodies, aggregation, etc..) well you may denature and linearize your protein and try to bind it again to the Ni resin. This will clarify if it is a kind of hidden tag issue...
Good luck...
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