I am purifying a 74 kDa protein (about half of the protein is predicted to be disordered) that has an His6-Maltose-binding protein tag (~118 kDa total) total. After eluting from the nickel beads I at first noticed precipitation when cleaving the His6-MBP tag during dialysis to remove the imidazole. However, when trying to purify the entire fusion protein I realized aggregation occurs as soon as I try to remove the imidazole, even if the tag remain on the protein. My dialysis buffer is 25 mM Tris buffer pH 7.4 that contains 400 M KCl, 10% glycerol, 1 mM DTT.
I have read adding EDTA (100 mM or so) to the dialysis buffer while trying to remove the imidazole can help. Any other suggestions?
The concentrated protein may require lower ionic strength or a different pH to prevent aggregation, or it may need to be less concentrated.
There are lots of things which influence protein aggregation. First off, how are you measuring the aggregation? Is your protein unfolding and forming non-specific blobs? Or is there a chance that it forms higher-order structures in the presence of your buffer? The latter is not a problem as many proteins form higher-order structures (not monomers). The former could be caused by a variety of factors: salt concentration, salt type, pH, temperature, protein concentration, presence (or lack) of additives. I presume your protein may have solubility issues since you are using an MBP tag in addition to the His tag for nickel affinity? Could your tag be interfering with folding? Are you getting soluble protein when you express? There are many factors to consider when trying to purify a well-folded, soluble protein.
I noticed that after removing the imidazole a white precipitate was visible. After dialysis I spun down the precipitate and analyzed the precipitate and soluble portion on an SDS-PAGE gel. Most of my protein was in the precipitate.
I actually began using the MBP tag because I noticed this boosted my protein's expression. Sounds odd, but it has worked for several members in my lab.
I do have soluble protein during expression, it is only after removing the imidazole that I observe the precipitation.
Is it necessary to remove the imidazole? What if you just leave it in?
I thought about that, however I have one assay that I will be performing in the future in which I cannot have imidazole.
When you do this assay, do you dilute the protein by a large factor? If so, you may be able to leave the imidazole in the stock solution because it will be diluted to a very low level in the assay, and the protein concentration will be so low that aggregation will not occur.
If this is not the case, perhaps you can replace the imidazole with some other small molecule that will have the same ability to keep the protein from aggregating, but will not interfere in the assay.
What happens to the imidazole requirement if you cleave off the His-MBP tag before starting the dialysis?
What happens if you leave out the 400 mM KCl from the dialysis buffer. That is a pretty high salt concentration?
100 mM EDTA seems like an unreasonably high concentration to add to the dialysis buffer. If the purpose is to chelate residual metals, you probably don't need more than 1 mM.
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