Abstract
AbstractMethotrexate‐resistant mosquito (Aedes albopictus) cell mutants were used as starting material for the purification of dihydrofolate reductase. Initial fractionation of a crude cell lysate by ammonium sulfate precipitation showed an enrichment of enzyme activity in the 60‐100% pellet. Purification of this ammonium sulfate fraction on a Sephadex G‐75 column prior to affinity chromatography was required for maximal yield of purified protein. A protein with dihydrofolate reductase activity and an Mr of 24,000 was eluted from a methotrexate‐agarose column with 0.5 M Tris‐HCl, pH 8.5, containing 2 mM folic acid. A 21‐fold increase in specific activity relative to that in the crude cell lysate was obtained, and amino acid sequence analysis confirmed that dihydrofolate reductase from the affinity column migrated as an electrophoretically pure band on SDS polyacrylamide gels. The initial 20 amino acid residues of the mosquito dihydrofolate reductase were identified by microsequencing and compared to those of murine, human, and bacterial dihydrofolate reductase molecules.
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