Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum

Abstract

The full-length pfdhfr-ts genes of the wild-type TM4/8.2 and the double mutant K1CB1 (C59R+S108N) from the genomic DNA of the corresponding Plasmodium falciparum parasite have been cloned into a modified pET(17b) plasmid and expressed in Escherichia coli BL21 (DE3) pLysS. Conditions for the expression and purification of the P. falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) have been established that yield ∼1 mg of the soluble active enzyme per litre of culture. The purified enzymes have been crystallized using a modified microbatch method with PEG 4000 as the primary precipitating agent. X-ray diffraction data were collected to 2.50 and 2.64 Å resolution under cryogenic conditions from single crystals of the two PfDHFR-TS proteins in complex with NADPH, dUMP and either Pyr30 or Pyr39. Preliminary X-ray analysis indicated that the crystals belong to the orthorhombic space group P212121, with two molecules per asymmetric unit and ∼52% solvent content (VM ≃ 2.6 Å3 Da-1). The use of a particular type of baby oil in the microbatch setup appeared to be beneficial to PfDHFR-TS crystallization and a preliminary comparison with another commonly used oil is described. © 2004 International Union of Crystallography. Printed in Denmark - all rights reserved.