Open AccessCharacterization, crystallization and preliminary X‐ray analysis of bifunctional dihydrofolate reductase–thymidylate synthase from Plasmodium falciparum
Author(s) -
Chitnumsub Penchit,
Yavaniyama Jirundon,
Vanichtanankul Jarunee,
Kamchonwongpaisan Sumalee,
Walkinshaw Malcolm D.,
Yuthavong Yongyuth
Publication year - 2004
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444904001544
Subject(s) - thymidylate synthase , plasmodium falciparum , dihydrofolate reductase , escherichia coli , chemistry , orthorhombic crystal system , enzyme , expression vector , microbiology and biotechnology , biochemistry , biology , crystallography , gene , crystal structure , recombinant dna , fluorouracil , genetics , chemotherapy , malaria , immunology
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 P 2 1 2 1 2 1 , with two molecules per asymmetric unit and ∼52% solvent content ( V M ≃ 2.6 Å 3 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.