Ni-purification blues again,

[Dima Klenchin]

svend at cajal.mbb.ki.se (Svend Kjaer) wrote:

:I have expressed substantial amounts of a receptor using a stable
:cell-line set-up.
:The receptor is his-tagged, which ideally should allow for facile
:purification using Ni-NTA beads added to concentrated serum-free
:conditioned medium.
:However, this is not the case. Each time I try to purify the protein, I
:get some back, but the majority passed through. From the same
:preparation, I have now tried three times and each time the same. The
:majority is in the flow-through and some is eluted specifically.
:I used a 50 mM Tris/150 mM NaCl, 1mM Imidazole buffer and wondered
:whether there are any suggestions to how to solve this problem - could
:the buffer be the reason - or could there be any compound in the
:conditioned medium, which competes with the his-tagged protein for
:binding ?

Could be a number of reasons.

- The tag is mostly buried in the fold and poorly accesible (check
binding under denaturing conditions). Just about the only remedy
is to change tag's placement. 
- The tag is getting cleaved by endogenous protease (check
bound and non-bound fractions) with anti His-tag Ab.
- The pH? For a reason still misterious to me, "my" protein binds
to Ni-NTA a lot better at 7.0 than it does at 8.0
- Your protein might be strongly negatively charged and with 
negative residual charge of Ni-NTA (I think?), it might repulse 
from the matrix. Increase your salt concentration to 0.5 M;
- Bind to the column, not in batch - weakly binding species 
are captured more efficiently in this format.

I had somewhat similar problems and while I cannot solve them
completely, I pushed the whole thing to a workable situation:
- bind to a column at 0 imidazole (10 mM Pi, 500 mM NaCl, pH 7.0),
wash extensively with the same;
- wash with 5 mM Imidazole (30 column volumes);
- wash with 10 mM imidazole (5 column volumes);
- elute with 100 mM imidazole (400 mM NaCl) - the protein elutes
at only 20 mM but in order to get it more concentrated, I bump
elution with higher, 100 mM;
- do a 40% ammonium sulfate cut of eluted fraction. 
- Result: >95% pure protein with about 75% yeild.

Hope this helps.

        - Dima