I have been expressing an intrinsically disordered protein (IDP) and have been having issues purifying it. The protein is overexpressed in T7 promoter E.coli cells. The protein has a 6His tag and was initially purified using a Nickel Column, but the contaminant protein -which is peptidyl propyl cis trans isomerase - was present at high levels. Using a cobalt column significantly improved the purification but could not eliminate the contaminant. A size exclusion purification step was added but the two protein come at the same peak so this step was not very good at purifying my IDP. Anion exchange did not help separate the two proteins because the interaction is hydrophobic and changing the pH or the salt concentration is damaging for the proteins as it degrades.
The buffer conditions used are: pH 8.0, 100mM tris HCl, 0.5M NaCl, 50 mM Arg/Glu.
Tried different low temperatures for expression because the protein is stressing the cells, however, the protein is still bound to the contaminant. Going lower, the cells will stop growing.
Anyone who has expressed IDP previously and struggled with contaminant proteins that were difficult to get rid of? What strategies helped you? Will using a lower pH help with stability of the IDP and reduce the co-purified protein?
Thank you for your help.
Hi Anca,
a couple of years ago I had to purify 4 intrinsically disordered proteins on behalf of a customer. They expressed at decent levels - as GST fusions - but when digested away from the tag carried some kind of chaperone with them that didn't come off when trying various published chaperone-removal tricks such as ATP-Mg2+ washes while the GST was bound to resin and so on.
I was lucky in one respect compared to your situation : the proteins were stable when I dialysed the salt concentration down to 50 or 100mM (I can't remember which), pH8. At this point, I thought I'd try performing IEX. One thing that I noted was that the chaperone had a much lower pI (around 4) than the proteins (around 6). As luck would have it, running the mixture of proteins through Q-sepharose resulted in the chaperone being left behind on the latter resin while the protein we wanted mostly ended up in the flowthrough, so I was able to achieve pretty effective separation.
Apart from Cobalt resin, have you tried Zinc? I use this a lot for some proteins and as with the advantage of Cobalt over Nickel, Zn can also give you cleaner purifications than the latter with some proteins.. If you don't have the resin itself available ready charged, take some Nickel or Cobalt resin, rinse with water, EDTA, water and then 100mM ZnSO4, followed by a bucket-load more water and then your binding buffer. I usually keep a bottle of uncharged chelating resin in the fridge in case of needing to try different ion combos for separation of tags, and also, recycled bulk NiNTA can be stripped and used for prototyping work comparing different resins with a protein to optimise purification.
Please feel free to discuss this further here. Good luck!
Robin
Have you tried hydrophobic interaction chromatography? What about selective salting-out precipitation? You can use ammonium sulphate, sodium sulphate or polyethylene glycol (PEG) as precipitating agents.
Looks like the isomerase is bound to your protein, which is not too surprising as it is supposed to help unfolded proteins fold, and IDPs are just that. As Robin suggested, the solution is to bind on of the partners to a column and then change the conditions so that the complex dissociates (that is, that the interaction of one partner with the column becomes stronger than the interaction of the two proteins).
Anca, have you tried a denaturing buffer (8 M Urea or 6 M Gdn plus your other buffer components)? Unless your IDP has an ordered domain that refolds poorly, this should have little effect on your protein of interest but may disrupt the interaction with the isomerase.
Thank you all for your suggestions
I have tried denaturing my protein but that just degrades it.
Robin Dickinson and Engelbert Buxbaum : I haven't tried Zinc column, I have already started looking into it, thank you. Changing salt conditions just precipitates my protein so I cannot use this. However, adding 5mM of ATP to my buffer helped with my chaperone.
Alemu Tekewe : I haven't looked into hydrophobic interaction chromatography, but changing the conditions of my buffers and adding a GST-tag helped with my purification.
Jordan T White: I cannot use a denaturing buffer as the protein is very unstable and following this step I cannot refold my protein.
Very glad to hear that you have found that ATP helps. With protein work it is very rare to have one miraculous change in conditions - small incremental improvements are much more commonplace. Good luck with the next steps.
Above all : keep smiling - a frown is a well known precipitant :)
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