Purification and characterization of dihydrofolate reductase of Plasmodium falciparum expressed by a synthetic gene in Escherichia coli.
We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase complex of P. falciparum in Escherichia coli, by constructing a gene with synthetic oligonucleotides that changed the gene's codon usages. The induced expression in an E. coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein. The product was precipitated in an inclusion body in a cell. Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl. Recombinant DHFRs with Ser or Thr at position 108 were prepared. Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108.
Sano
G
G
Department of Protozoology and Parasitology, Osaka University, Japan.
Morimatsu
K
K
Horii
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015195:Drug Design; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D005813:Genes, Synthetic / Q000235:genetics*; D005815:Genetic Code; D002479:Inclusion Bodies; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011489:Protein Denaturation; D011739:Pyrimethamine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D014295:Trimethoprim / Q000494:pharmacology
D004793:Enzyme Reactivators; D011994:Recombinant Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate; D011739:Pyrimethamine