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
