pGEX fusions - GST band appearing.
as at macpost.pharmakologie.uni-erlangen.de
Wed Dec 27 05:55:13 EST 1995
In article <1995Dec18.162454 at molbiol.ox.ac.uk>, rpgrant at molbiol.ox.ac.uk
(Mad Dan Eccles) wrote:
> I think we've done this.... but...
> Members of the lab have been getting band(s) around 28 kDa on their gels when
> doing GST-fusion protein preps. The occurence of these bands varies,
> they're there, sometimes not. There is a reaction at 28 kDa with
> it looks like the GST fusion partner.
> My feeling is that it is premature termination of translation, but I have not
> yet done a Western to tell me whether this is so.
> Does this ring any bells with anyone?
Yes it does!
We have at present similar problems in that eluted proteins give at first
the expected size of the fusion protein but after a couple of days in the
cold room the separate domains appear. GST at around 26 kDa and the fused
protein at 20-22 kDa depending on the construct. The cleavage seems to be
100% as detected by Western blotting. It looks like Thrombin cleavage
although the preparations have never seen Thrombin - only protease
This has happened several times now and in all cases it is *specific*
cleavage and not general breakdown or digestion by contaminating
bacteria/moulds or the like. - Looks as if we could save the money for
Thrombin in the future.
We have no explanation for this at the moment.
Has anybody else?
Axel Schumacher, Ph.D. ! Phone:+49 9131 852002
Institute for Pharmacology ! FAX: +49 9131 206119
University of Erlangen !
D-91054 Erlangen Germany ! as at macpost.pharmakologie.uni-erlangen.de
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