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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