Dear ABRFers,
I recently posted a message with respect to GST-6His-double fusions and I
was asked by several to mail all answers to the ABRF community in case they
would be sent to me directly.
OK, here's a short summary:
My question was: we constructed a double fusion consisting of a GST part
followed by a hexa-histidine sequence fused to the N-terminus of a protein.
Both tags seem to be OK, since the protein can be easily purified on
glutathione as well as Ni-NTA columns. The intention behind this was that
neither of the two purifications yielded a pure preparation. So we tried to
combine both tags by first going Ni-NTA, eluting with imidazole, and taking
the eluate and loading it onto a glutathione column. We didn't expect
difficulties, but after the Ni-NTA step, the protein failed to bind to the
glutathione column. Why is this?
One reply was:
>
>If imidazole does interfere with glutathione binding, I have used EDTA or
>EGTA to elute proteins off Ni-NTA before to avoid imidazole in the product.
>The result certainly wasn't as clean as the more specific eluate but it was
>effective when coupled with a few high salt/detergent containing washes.
>
Good suggestion!
Next reply:
>
>It could be possible that you have a small amount of interfering Ni in
>your sample after eluting from Ni-NTA. 0.5 M is a high concentration for
>>imidazole (a strong eluting agent for IMAC) and it could be possible to
>unbound >a small amount of Ni.Some authors recommend to saturate the
>column with >imidazole (low concentration) in the starting buffer prior to
>adsorption of the >protein sample, when imidazole will be used for elution.
>If this doesn't work I recommend you to change the buffer (G-25 or
>similar) after chromatography in Ni-NTA to eliminate the possible
>interference >of Ni.
>
Next reply:
>
>Are you doing a thorough buffer exchange after the first elution? Ionic
>strength might affect binding, even with an affinity tag.
>
No, we didn't. As far as I know, binding of GST to glutathione can tolerate
salt up to 0.5 M (at least NaCl). But it could be different with imidazole.
Next reply:
>
>You want to purify His/GST-tagged protein. (if tags are separeted into N
>and C >terminus,better to purify :-) L-Histidine also works as the eluent.
>(but I don`t know whether it also works well to purify GST-tag )
>I found this information shown below, you can also search easily
>(here also shown L-his works better than EDTA.)
>http://tto.biomednet.com/
>As alternative way to remove imidazole from 1st eluted sample,
>how about using buffer exchange by dialysis ???
>And after that, try to purify, if needed.
>In our lab, my colleage constructed
>NH2- GST - [ORF] -HA-His-COOH.
>In this case, he could purify enough only by Ni-NTA.
>
just a quick comment to this e-mail: yes, I certainly agree to separate the
tags into N-terminal and C-terminal ends. However, whatever we did to the
C-terminus of this protein, it lost its function (it's an extremely
sensitive kinase). We can fuse tags only to the N-terminus.
Next reply (was in german, so I'm translating, but Peter thanks a lot for
the answer!):
it was suspected that Ni was coeluting with the protein, and therefore
poisoning the glutathione resin. The suggestion was to remove Ni with EDTA.
Good suggestion as well!
Next reply:
>
>My first inclination would be to add DTT (5-10 mM) back to the protein
>after >the Ni column. The GST and glutathione need to be
>reduced for good >interaction and a Ni column would, if anything, be
>oxidizing.
>
Will be tried out!
Next reply:
>
>Try dialysis of the protein against the appropriate buffer between each
>affinity chromatography step. We also dialyse before addition to the Ni-NTA
>column if this is the first step.
>
Well, we tried dialysis and lost the protein totally! But I am sure this
could be improved. It just seems to be a sticky protein and if we would
include all the goodies necessary, I am sure it could be dialyzed. But
still, one would have to optimize. Also, this protein is a real bugger. For
each experiment we need to grow five liters of yeast culture. You don't
want to grow all this stuff just to find out how to dialyze!
Thank you very much for all ABRFers who responded! We have now a couple of
good suggestions to try out. If the community is interested, I can send a
follow-up message on the outcome of this low-tech double tag!
Best regards
Paul
Paul Jenoe, PhD
Department of Biochemistry
Biozentrum of the University of Basel
Klingelbergstrasse 70
CH-4056 Basel
Switzerland
Tel. +41 61 267 21 57
Fax. +41 61 267 21 48
e-mail Paul.Jenoe@unibas.ch
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