Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Base Sequence
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Drug Design
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Enzyme Reactivators
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Escherichia coli / genetics
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Genes, Protozoan / genetics*
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Genes, Synthetic / genetics*
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Genetic Code
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Inclusion Bodies
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Methotrexate / pharmacology
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Molecular Sequence Data
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Plasmodium falciparum / enzymology*
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Plasmodium falciparum / genetics
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Protein Denaturation
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Pyrimethamine / pharmacology
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Recombinant Proteins / biosynthesis
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Tetrahydrofolate Dehydrogenase / biosynthesis*
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Tetrahydrofolate Dehydrogenase / drug effects
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Tetrahydrofolate Dehydrogenase / genetics
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Trimethoprim / pharmacology
Substances
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Enzyme Reactivators
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Recombinant Proteins
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Trimethoprim
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Tetrahydrofolate Dehydrogenase
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Methotrexate
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Pyrimethamine