The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants.
A 693-base pair gene coding for the 27,132-dalton dihydrofolate reductase (DHFR) domain of the thymidylate synthase-dihydrofolate reductase (TS-DHFR) bifunctional protein of Plasmodium falciparum was designed to have Escherichia coli codon preference and multiple unique restriction sites and was chemically synthesized. The gene was overexpressed (> 50% total cellular protein) in E. coli as insoluble inclusion bodies which could be unfolded and refolded to recover soluble enzyme activity. The refolded DHFR was purified by methotrexate-Sepharose affinity chromatography to give the homogeneous enzyme. Active site titration with methotrexate revealed that the purified protein was fully active. The purified DHFR migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 30 kDa, and gel filtration showed that the protein is a monomer. The yield of purified enzyme was about 5-6 mg/liter of bacterial culture. Kinetic properties of the purified recombinant DHFR were similar to those reported for wild type bifunctional TS-DHFR. Cassette mutagenesis of the synthetic gene was performed to give the S108N and the N51I + S108N mutants which provided DHFRs analogous to pyrimethamine-resistant mutants found in nature.
Sirawaraporn
W
W
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Prapunwattana
P
P
Sirawaraporn
R
R
Yuthavong
Y
Y
Santi
D V
DV
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli; D005813:Genes, Synthetic; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology*; D017510:Protein Folding; D011739:Pyrimethamine / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013940:Thymidylate Synthase / Q000737:chemistry*
D004274:DNA, Recombinant; D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008727:Methotrexate; D011739:Pyrimethamine