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Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum
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Document Title
Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum
Author
Chitnumsub P., Yavaniyama J., Vanichtanankul J., Kamchonwongpaisan S., Walkinshaw M.D., Yuthavong Y.
Name from Authors Collection
Affiliations
BIOTEC, National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, United Kingdom
Type
Article
Source Title
Acta Crystallographica Section D: Biological Crystallography
ISSN
09074449
Year
2004
Volume
60
Issue
4
Page
780-783
Open Access
Hybrid Gold
DOI
10.1107/S0907444904001544
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.
License
CC BY
Rights
Author
Publication Source
Scopus