DHFR and methotrexate

[Thomas O'Neill]

On 13 Mar 1995 jeg at PW.USDA.GOV wrote:

> We need advice from some kind person with experience in purifying DHFR 
> (dihydrofolate reductase) fusion proteins on methotrexate affinity 
> columns.  We've expressed our peptide of interest as a DHFR fusion.  Our 
> problem is that when we apply it to the methotrexate (MTX on CNBr 
> activated agarose beads, Sigma) it seems to bind well but only about 20% 
> comes off again in the elution buffer( 3 mM folate, 0.5M KCl, 10 mM 
> KPhosphate, 0.2 mM EDTA, 0.1 mM DTT).  We're aware that MTX columns have 
> problems with irreversible binding and tried pre-treating with an 
> extract of E. coli protein but it didn't help. Could someone please tell 
> us what kind of recoveries are expected from MTX columns?  We're 
> thinking of adding a His tag to the amino end of the DHFR and using a Ni 
> column for purification.  Would this be likely to give better yields?  
> Thanks for any help- Joan  
> 
> 
  Though I've never purified DHFR fusion proteins on MTX affinity 
columns, I have successfully purified a hell of a lot DHFR from 
over-expressing mammalian cell lines. I also used the Sigma resin, but I 
always eluted with either reduced folic acid or methotrexate itself. My 
recoveries were good. The biggest problem I encountered was what 
appeared to be leaching of the methotrexate into the column over time. 
Thorough washing of the column prior to applying your protein is all that 
is neccessary.
  There was a protocol that the lab used to make reduced folate that was 
really simple. It was an acid/base titration in the presense of some 
reducing agent whose name escapes me. Unfortunately for you, and 
fortunately for me, I no longer work in that lab, so I can't give you a 
reference. It was an OLD paper, like 1970's or something.

					Good luck,
					Tom O'Neill