Janice, I talked to a friend of mine and she suggested the following:
_______________________________________________________________________________
Hi, Kristine,
Thanks for the message. It is always great to hear from you. This
observation is quite interesting. I wonder if the reason for the GroEL
binding to the glutahtione column might be that it binds to the
unfolded or partially unfolded GST-fusion protein. If this is true,
than Mg-ATP should release groEL from this protein.
Best regards,
Maria
_______________________________________________________________________________
Hope that helps, Kristine
Kristine Swiderek
Assoc. Research Director, Biological Structure
ZymoGenetics, Inc.
1201 Eastlake Ave. E
Seattle, WA 98102
Phone: (206)515-4901
Fax: (206)442-6608
e-mail: swiderek@zgi.com
_______________________________________________________________________________
Subject: GST Fusion Proteins
From: "Bleibaum; Janice (USA;Palo Alto)" <JANICE.BLEIBAUM@roche.com> at
smtpgate-zgi
Date: 5/22/98 2:18
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
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From: "Bleibaum, Janice (USA;Palo Alto)" <JANICE.BLEIBAUM@roche.com>
Subject: GST Fusion Proteins
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