Hi,
Common issue indeed (a score of E. coli proteins have high affinity to IMAC
media). I've never seen this particular protein as a contaminant, but then
again I do not always identify all the contaminants as long as I can get rid
of them :)
Things to try:
0. if you really suspect a specific interaction with your protein - then the
thing to check is a 'blank' E. coli extract loaded and eluted in the same
conditions. Does the offending contaminant not bind in the absence of your
target protein ? If you discover that the interaction is specific then you
have to experiment - salts, detergents, polyols, NDSB's - this kind of stuff
can help you dissociate the two. Even low concentrations of urea or
guanidine hydrochloride - anything goes at that stage.
1. different media and metal - Ni-NTA, Co-NTA, His-SELECT (Ni), His-SELECT
(Co), TALON, HisTrap, etc. Depending on the resin you would need to elute
with varying amounts of imidazole so your best bet is to always elute with a
gradient at first.
2. As suggested by others - HIC is a very useful and frequently neglected
method of separation.
3. Good old fashined AS cut.
4. A glucosamine column or something similar (heparin column?) - it may suck
out the contaminant based on affinity.
5. Cleaveable His-tag, as suggested earlier.
6. treat the protein mixture with one of the native ligands (just like ATP
and GroEL/S
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Ailong
Ke
Sent: Friday, November 02, 2007 2:43 PM
To: [log in to unmask]
Subject: [ccp4bb] Ni-NTA purifications contaminated with E coli
Glucosamine-fructose-6-phosphate aminotransferase
>Hello,
We are trying to purify an N-terminal His6-tagged protein from E.
coli, and the prep was contaminated with the E coli
Glucosamine-fructose-6-phosphate aminotransferase, which co-purifies
with my protein of interest in subsequent ion-exchange and sizing
columns. This protein appears to be a common contamination in the
Ni-NTA purifications. Does anyone have tricks to get rid of it?
Thanks.
Ailong
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