Paul,
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.
God luck...Ken
> -----Original Message-----
> From: jenoe@ubaclu.unibas.ch [mailto:jenoe@ubaclu.unibas.ch]
> Sent: Wednesday, January 17, 2001 7:04 PM
> To: Recipients of ABRF List
> Cc: Paul Jenoe
> Subject: Hexa-His/GST-tag
>
>
> Dear Colleagues,
>
> we've spent a considerable amount of time to put a double
> handle onto a
> yeast protein which is expressed in exceedingly low amounts.
>
> First, we put the gene under the control of the Gal promoter,
> and we added
> two affinity handles to the N-terminus for easy purification:
> a GST tag
> followed by a 6-His tag. We carefully checked the protein for
> functionality in a yeast complementation assay, and it is
> functional. We
> can purify the protein by passing an extract over Ni-NTA
> columns, or over
> a glutathion column. Although the protein is not pure by either of the
> steps alone, the tags work nicely. But when we want to link both
> purification steps together to purify the protein to homogeneity, the
> approach doesn't work. If we go via Ni-NTA first, we cannot retain the
> protein on the glutathion column, when we go glutathion
> column first, we
> cannot get it to stick to the Ni-NTA column any more. Both columns are
> eluted in the usual manner: Ni-NTA with 0.5 M imidazole in
> PBS, buffered
> to pH 7.5, the glutathion column with 10 mM glutathion. So
> all criteria
> for binding are fulfilled. I know that Ni-NTA is sensitive to reducing
> agents, this is why we go Ni-NTA first, followed by a
> glutathion column.
> But that binding via the GST part would be influenced by imidazole, I
> doubt. Does anybody have experience with double tags (of course others
> than the en vogue TAP stuff). Any input is highly appreciated.
>
> Best regards
>
> Paul Jenoe, PhD
> Department of Biochemistry
> Biozentrum of the University of Basel
> Switzerland
> Phone ++41 61 267 21 57
> Fax ++41 61 267 21 48
> e-mail Paul.Jenoe@unibas.ch
>
>
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