Publication: Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum
Issued Date
2004-04-01
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ISSN
09074449
Other identifier(s)
2-s2.0-11144285073
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Mahidol University
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SCOPUS
Bibliographic Citation
Acta Crystallographica Section D: Biological Crystallography. Vol.60, No.4 (2004), 780-783
Suggested Citation
Penchit Chitnumsub, Jirundon Yavaniyama, Jarunee Vanichtanankul, Sumalee Kamchonwongpaisan, Malcolm D. Walkinshaw, Yongyuth Yuthavong Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum. Acta Crystallographica Section D: Biological Crystallography. Vol.60, No.4 (2004), 780-783. doi:10.1107/S0907444904001544 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/21206
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Title
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.