imidazol eluent alternative in his-tagged proteins

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Nov 1 14:35:56 EST 1999



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 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.
>
> Thanks a lot for any hint or comment
> Soenke

Hi Soenke,
Try elution with low pH. It works great and does not usually harm your
protein. I routinely use solutions buffered at pH 6.3, pH 5.5, pH 4.8
(e.g. phosphate buffer). Most proteins elute at 5.5, some at 4.8. It is
usually desirable to back-titrate to neutral as soon as possible.
There are two potential problems:
1. The protein does not elute. Try 50mM HCl as eluent, and allow to
elute directly into a reaction tube containing 1M Tris, pH 8.0.
2. The protein gets "degraded". If you see distinct degradation bands,
your protein may be susceptible to acid hydrolysis. This kind of
degradation is enzyme-independent (so protease inhibitors won´t change
the situation), and usually cleaves proteins at DP bonds (the peptide
bond between aspartic acid and proline). In that case, acid elution is
NOT an option, of course.

A second possible way to elute your protein is to chelate the nickel
with EDTA. Try elution with 100mM EDTA. The column (which is usually
slightly blue) will turn white as the nickel elutes together with your
protein. Elution with EDTA also works very well, but has the drawback
that the eluted protein will have to be dialysed to get rid of nickel
and EDTA.

As to cleavage from the column: This works if you don´t need lots of
your protein. You can e.g. use enterokinase, factor Xa, or thrombin, but
these enzymes are quite expensive. So for larger amounts of recombinant
protein it is not economical to do. In addition, the commercial enzymes
are not very pure. This means you cleave off the protein and at the same
time contaminate it with the protease (and the other proteins in the
enzyme preparation). I wouldn´t resort to that unless absolutely
necessary.

Hope this helps,
Gruesse aus Konstanz,
Frank





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