BIOMAT Database Logo BIOMAT Database Logo

The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants. 1993

W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008727 Methotrexate An antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of TETRAHYDROFOLATE DEHYDROGENASE and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA. Amethopterin,Methotrexate Hydrate,Methotrexate Sodium,Methotrexate, (D)-Isomer,Methotrexate, (DL)-Isomer,Methotrexate, Dicesium Salt,Methotrexate, Disodium Salt,Methotrexate, Sodium Salt,Mexate,Dicesium Salt Methotrexate,Hydrate, Methotrexate,Sodium, Methotrexate
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009097 Multienzyme Complexes Systems of enzymes which function sequentially by catalyzing consecutive reactions linked by common metabolic intermediates. They may involve simply a transfer of water molecules or hydrogen atoms and may be associated with large supramolecular structures such as MITOCHONDRIA or RIBOSOMES. Complexes, Multienzyme
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010963 Plasmodium falciparum A species of protozoa that is the causal agent of falciparum malaria (MALARIA, FALCIPARUM). It is most prevalent in the tropics and subtropics. Plasmodium falciparums,falciparums, Plasmodium
D011739 Pyrimethamine One of the FOLIC ACID ANTAGONISTS that is used as an antimalarial or with a sulfonamide to treat toxoplasmosis. Chloridin,Daraprim,Malocide,Tindurine
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004274 DNA, Recombinant Biologically active DNA which has been formed by the in vitro joining of segments of DNA from different sources. It includes the recombination joint or edge of a heteroduplex region where two recombining DNA molecules are connected. Genes, Spliced,Recombinant DNA,Spliced Gene,Recombinant DNA Research,Recombination Joint,DNA Research, Recombinant,Gene, Spliced,Joint, Recombination,Research, Recombinant DNA,Spliced Genes
D004351 Drug Resistance Diminished or failed response of an organism, disease or tissue to the intended effectiveness of a chemical or drug. It should be differentiated from DRUG TOLERANCE which is the progressive diminution of the susceptibility of a human or animal to the effects of a drug, as a result of continued administration. Resistance, Drug

Related Publications

W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase.
June 1997, Biochemical and biophysical research communications,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum.
December 1990, Biochemistry,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Study of dihydrofolate reductase-thymidylate synthase in Plasmodium falciparum.
October 1988, The American journal of tropical medicine and hygiene,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
[Gene point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum].
January 2001, Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase.
January 2005, Molecular and biochemical parasitology,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase.
February 1997, Proceedings of the National Academy of Sciences of the United States of America,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Studies of Plasmodium falciparum dihydrofolate-reductase thymidylate synthase: potential advantages.
January 1989, Progress in clinical and biological research,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.
December 1987, Proceedings of the National Academy of Sciences of the United States of America,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.
June 2012, Parasitology international,
W Sirawaraporn, and P Prapunwattana, and R Sirawaraporn, and Y Yuthavong, and D V Santi
Cell-free production of functional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase.
February 2007, Molecular and biochemical parasitology,
Copied contents to your clipboard!