pKa of a His tag.
kellere at hotmail.com
Mon Feb 18 01:21:29 EST 2002
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
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:
Succinate | Arginine*
NaH2PO4 | Na2HPO4*
* tested with NaCl concentrations of 100 mM, 200 mM and 300mM
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