Having got some pretty useful tips already from networkland, I'm
wondering if anyone out there has had any experience of using tagging
systems for protein purification, such as the Histidine tag purified on
NTA resin, or the FLAG fusion system marketed by IBI where the fusion
protein is affinity-purified on an antibody column. We are looking for a
suitable method to purify variants of a protein, constructed by directed
mutagenesis, in a cell where the wild type protein is also present (it has
to be as its essential for viability and the mutant proteins may well be
inactive). Also, the protein needs to be kept in
its native form throughout the purification if possible. Making the mutant
forms with a tag on that allows them to be separated from the wild type seems
a suitable route to try. Anyone out there had a go with this? Does the
presence of the tag tend to have a major effect on activity, and is there
any a priori reason for having it at the N or C terminus? (This
is a cytoplasmic protein so we're not interested in trying to secrete it).
Just how valid are the claims of IBI and Qiagen that you can get good one
step purification? Etc, etc, etc. All reponses gratefully received, and
I'll post a summary.
(This is the second attempt to post this message; apologies to all concerned
if it appears twice!)
Many thanks,
Pete Lund
Biological Sciences
University of Birmingham
Birmingham B15 2TT
UK
LUNDPA at UK.AC.BHAM.VAX1
