DHFR and methotrexate

Barry Schweitzer barry at fhis.net
Wed Mar 22 16:01:17 EST 1995


Thomas O'Neill (toneil1 at gl.umbc.edu) wrote:
: 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 
The reducing agent is dithionate and the reference is Blakley, R. L. (1960)
Nature 188, 231-232.  I agree with Tom, 200 uM dihydrofolate may work better
than folic acid.  Also make sure that your elution buffer is around pH 8.
The more acidic the pH, the tighter MTX binds to DHFR.

Good luck!

--
Barry Schweitzer, Ph.D.
Director                                     Assistant Professor
Division of Structural Biology               Department of Chemistry
Walt Disney Memorial Cancer Institute        University of Central Florida
 at Florida Hospital
12722 Research Parkway
Orlando, FL 32826
Phone:	(407) 380-9977
FAX:	(407) 380-9978
email:	barry at sneezy.fhis.net



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