Ni-NTA column and internal poly-His stretch.

Orhan owe1 at le.ac.uk
Fri Sep 29 03:22:17 EST 1995


In article <DFMwJu.527 at cc.umontreal.ca>, daniel durocher
<daniel_durocher at IRCM.UMontreal.CA> wrote:

> Hello again everybody!
> 
> I am working with a transcription factor that is present in very low
> abundance. It possess a heptahistidine stretch in a region predicted to 
> be at the surface. I would like to semi-purify it by Ni-NTA agarose 
> affinity chromatography.
> 
> Is it feasible? (I want to work in native conditions).
> 
> Thanks!

Depends on several things. First off it would help if the heptahistidine
stretch is a concurrent heptahistidine stretch and not 7 histidines fairly
close together in a region separated by other residues. This might not
necessarily present residues closely enough to cause a strong interaction
with the IMAC column.  Second, as it's the imidazole side chain that form
bonds with the Ni atom they have to be exposed, if the pH environment of
the protein is high enough in vivo, the imidazoles will be de-protonated
hence pretty hydrophobic in nature and may be buried even though the
stretch of backbone on which they appear is predicted as being on the
surface. 

Don't know if this really helps much. Potentially it may work but it
depends on the structure and environment of the protein. Try it and see
*8).

Orhan.

-- 
Orhan W.D. Ertughrul         "Its hard enough to watch the news let
Dept. of Biochemistry         alone explain it to a child to cast
Leicester University          its eyes cross nature over fields of      
University Road               rape and corn and tell it without 
LE1 7RH                       flinching not to fear where it's       
                              been born."
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