pKa of a His tag.

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Mon Feb 18 11:34:10 EST 2002


Normally, IIRC, at ph 7 histidines are neutral. So, you can be right or you
could be wrong. Until you purify your protein so that your final product
does not have a his-tag, you won't know for sure :) You can try cutting the
tag off with that weird specific carboxypeptidase that needs 37 c to work
(anyone remember its name ?) or you could try engineering a traditional
cleavage (TEV, etc) site just before the tag.

You may discover that your protein just does not like to pass pH 7, his-tag
or no his tag. You may also discover that if you plunge your protein into
say, pH 4.5 it may stay up. Why do you need pH 7, anyway ? To slow down N-H
exchange ?

A.G.E.

"John Everett" <kellere at hotmail.com> wrote in message
news:5a7ca595.0202172221.298d5fbb at posting.google.com...
> Newsgroup...
>
>   Placing a His tag onto a target protein is one
>   of my favorite methods of purifying a recombinant
>   protein. But I believe that a His tag has betrayed
>   me. I am trying to lower the pH of my his tagged
>   protein from pH 7.5 to 7.0.  As I approach pH 7.0
>   the protein ( pI of 5.5 ) crashes out of solution.
>   The protein possesses several negatively charged
>   residues that cluster in sequence and in homology
>   models. Since the his tag is on the carboxy terminal
>   and adjacent to the carboxy terminal end of a
>   predicted helix... I believe that the pKa of the
>   Histidines in the His tag would be greater than 6.5.
>   If this is true,  then when the protein approaches a
>   pH of 7.0, the Histidines in the His tag would become
>   protonated, thus charged, creating a large cluster
>   of possitive charge. This positive charge  may in turn
>   bind to other protein molecules (since the protein is
>   so negatively charged) and cause it to fall out of solution.
>
>   All of this effort to drop .5 pH units is to create
>   an NMR sample. A pH of 6.8 would be great but pH 7.0
>   will do.
>
>   Any thoughts on the validity of this argument
>   or information about the pKa of a carboxy terminal
>   His tag would be greatly appreciated.
>
>   buffers already explored without success:
>
>   Tris-HCl*
>   Bis-Tris*
>   HEPES*
>   MOPS*
>   PIPES*
>   Succinate | Arginine*
>   NaH2PO4   | Na2HPO4*
>
> * tested with NaCl concentrations of 100 mM, 200 mM and 300mM
>
>   Thanks!
>       John Everett





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