Hi everyone
I have a problem with Histag protein purification. I have this construct with his tag on N-terminal. I went for small-scale expression and I had some good bands. However, on a large scale, the protein didn't bind with the HiTrap TALON cobalt. My protein contains an Mg2+ ion coordinated by the side chains of Asp, His. How to overcome this metal ion issue, or Should I change the column to a Ni-Trap?
I appreciate your help
I doubt the Mg2+ in the active site is relevant here. Do you have any chelators or metal ions in the sample before it goes onto the column? When you say the protein does not bind, do you really mean it doesn’t bind or are you saying you didn’t manage to elute anything? I think it’s important to know whether you have a binding or elution issue. Either way you’ll end up with no protein. Do you have confirmation of successful expression on SDS gels?
Nick Gee Sorry for the weak explanation. It's a binding issue, the proteins came out in the flow throw, and the SDS gel confirms this.
Since my protein contains 2 metal binding sites that also involve histidine I was curious if this would be the reason.
in the attached, the histag is referred to in yellow color. the metal binding sites in blue and red.
Dear Aymane El Fellah
It may happen that HisTag is buried in the protein and, thus, unavailable for the binding group on a column. You can load a protein on a column in a solution with urea or guanidine hydrochloride to unfold a protein. Then, you can refold a protein on a column and elute it.
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