DHFR and methotrexate

jeg at PW.USDA.GOV jeg at PW.USDA.GOV
Mon Mar 13 12:21:11 EST 1995


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  



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