I've seen this phenomenon before. You might suggest adding ATP
and Mg. In our hands, these additions allowed some (not all) of our
protein to be kicked out of the GroEL complex.
If they don't need their protein in a native conformation,
detergents worked very well to release our protein, as did urea.
I suspect that, at least in our case, the protein was not folding
correctly (probably inside out with the hydrophobics on the surface) so
that it was getting stuck in the folding pocket of GroEL.
Hope this helps,
Jeanne
On Fri, 22 May 1998, Bleibaum, Janice (USA;Palo Alto) wrote:
> Hello,
>
> A big, hearty, thank you (!) to all who responded to my message
> regarding service from PerSeptive Biosystems. Things are progressing
> this week and we will hopefully have the instrument spec'd out by the
> middle of next week.
>
> My question today concerns GST fusion proteins. A user is trying to
> purify a GST fusion protein (prior to thrombin cleavage) which runs at
> 70kD on SDS-PAGE. Although the GST seems partially blocked, I can
> identify his 70kD band as beginning with GST. The major protein coming
> off his glutathione column, however, is chaperonin 60 (groEL) which runs
> at 60kD on SDS-PAGE. As a rough guess, I'd say there's four times as
> much groEL as the desired GST fusion protein. I have seen something
> very like this before. Some years ago, I sequenced a
> glutathione-purified "GST fusion protein" which back then I only recall
> identifying as a heat shock protein; I don't remember if it was groEL or
> not (or if groEL was as well characterized then).
>
> It seems that groEL is binding glutathione. I was wondering if anyone
> has seen this phenomenon and if there are any quick/easy tricks to get
> rid of the groEL. I imagine that, if an antibody against groEL were
> available, that one could immunoprecipitate the groEL but I don't know
> how well this would work. All ideas welcome.
>
> Thanks!
>
> Janice
>
> Janice Bleibaum
> Protein Sequencing Laboratory
> Roche Bioscience
> 3401 Hillview Ave.
> Palo Alto CA 94304
> (650)852-1639
> (650)354-7554FAX
> janice.bleibaum@roche.com
>
>
****************************************
L. Jeanne Perry, Ph.D.
Molecular Biology Institute, Rm 113
Box 951570
University of California, Los Angeles
Los Angeles, CA 90095-1570
(310)206-2871 FAX: (310)206-3914
perry@ewald.mbi.ucla.edu
****************************************