pKa of a His tag.

[John Everett]

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