imidazol eluent alternative in his-tagged proteins

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Nov 1 16:59:01 EST 1999


behrends at plexus.uke.uni-hamburg.de (Soenke Behrends) wrote:
:
:Dear netters,
:
:I am working on a hemoprotein where the heme is bound
:to the protein by a histidin (and specifically by the imidazol
:moiety). My impression and fear is, that after his-tag binding
:to a Ni column the elution with imidazol buffer not only 
:destroys the protein column interaction but also frees 
:my protein of heme. Does anyone know of an alternative
:way of elution (under native conditions) get the protein
:off the column. 

I doubt it! If you are indeed concerned do the following. 
Add 250 mM imidazole to your protein solution, wait an hour
or so, than do spin gel-filtration over Sephadex G-25 or Bio-Gel
P6 (such columns are sold commercially or can be easily 
prepared in the lab). If eluate (excluded protein) is colorless, 
then you do have heme dissociation. If the protein is still 
colored, you don't have anything to worry about. 
(or simply measure heme's absorbance shift which usually is 
pretty big). 

In any case, you can always try elution at pH < 6.0
if your protein survives it (not all do). 

Finally, there is always an option to wash the column with
20 mM imidazole and elute bound protein by 50 mM EDTA
(then dialysis to get rid of Ni2+/EDTA).

:I have thought also to construct a  His-tag with TEV protease
:site and cleave it off the column, but I am forced to tag
:the C-terminus and I have not found any vector / technique
:to do that. 

You always have an option to insert the tag manually at C-terminus
in frame with the upsteam sequence. In most cases this requires
blunt end ligations but that's no problem, right? 

        - Dima




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