Characterization, crystallization and preliminary X-ray analysis of bifunctional dihydrofolate reductase-thymidylate synthase from Plasmodium falciparum.
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 approximately 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 A 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 P2(1)2(1)2(1), with two molecules per asymmetric unit and approximately 52% solvent content (VM approximately 2.6 A3 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.
Chitnumsub
Penchit
P
BIOTEC, National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand. penchit@biotec.or.th
Yuvaniyama
Jirundon
J
Yavaniyama
Jirundon
J
Vanichtanankul
Jarunee
J
Kamchonwongpaisan
Sumalee
S
Walkinshaw
Malcolm D
MD
Yuthavong
Yongyuth
Y
D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides; D005818:Genetic Engineering; D009249:NADP; D010963:Plasmodium falciparum / Q000201:enzymology*; D011092:Polyethylene Glycols; D015800:Protozoan Proteins / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013940:Thymidylate Synthase / Q000737:chemistry*
D003856:Deoxyuracil Nucleotides; D015800:Protozoan Proteins; D011092:Polyethylene Glycols; D009249:NADP; C007267:2'-deoxyuridylic acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase