Hi
In addition to what Fred and others have said, it is important to remember
there are several native, stress-responsive proteins of E. coli that show
good affinity for metal ions. Hence, they can be easily co-purified by
immobilised metal affinity chromatography (IMAC). This seems particularly
critical when the recombinant protein/protein domain of interest is
expressed at very low levels and/or shows low metal binding capacity. A
number of reasons for this include having the histidine tag partially
buried, low intrinsic stability of your protein; its tendency to aggregate;
etc.).
We wrote a short review on this issue not long ago hoping it would help to
identify quickly the usual suspects (doi:10.1016/j.bbagen.2006.03.027).
I totally endorse what others have said: by all means always check the DNA
sequence of what you want to express, and confirm its identity once you have
purified it (N-terminal sequence, mass spec., etc.) to avoid disappointment
and save resources.
Kind regards
Victor
(Tom Blundell lab).
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
CB2 1GA
Cambridge
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Kerff
Frédéric
Sent: 18 February 2010 23:16
To: [log in to unmask]
Subject: Re: [ccp4bb] Expression of a protein of 43KDa
The maltose binding protein is one possibility. Some collaborators did
provide us with very pure MBP instead of the protein of interest...
Mass spec eventually identified it after we wasted some time on it...
Fred
> I am trying to overexpress a His-tagged protein of 29KDa in E.coli
> (BL21-codon plus) and I end up with a highly expressed product that of
> 43KDa that binds to the Ni-column. I also have nice crystals. Does
> anyone have any experience on this.
> Armando
>
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