Recent Advance on Thymidylate Synthase-Dihydrofolate Reductase from Plasmodium falciparum
The coding sequence of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from moderate pyrimethamine resistant strain of Plasmodium falciparum was assembled in a pUC expression vector. Insertion of a synthetic oligonucleotide duplex containing a ribosomal binding sequence at 5’ upstream to ATG start codon resulted in a recombinant plasmid which expressed catalytically active TS-DHFR in E. coli. The expression of Plasmodium enzyme was demonstrated as follows: First, transformation of a TS-deficient E. coli resulted in genetic complementation of the deficiency and the activities of TS and DHFR were significantly higher than those in control host extracts. Second, TS and DHFR activities copurified upon chromatography with an apparent molecular weight ∼ 110 kDa, a characteristic of protozoan bifunctional enzyme. Third, the enzyme formed a covalent complex with [3H] FdUMP-CH2H4folate, and SDS polyacrylamide gel electrophoresis showed the subunit of the recombinant enzyme to be 67 kDa. Finally, N-terminus sequencing of the 67 kDa protein indicated that the N-terminus was not blocked and the sequences of the first 10 amino acids perfectly matched the reported nucleotide sequence. The recombinant enzyme was purified to homogeneity using 10 formylfolate affinity chromatography followed by FPLC on Mono Q column. Since the coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include point mutations thus far linked to pyrimethamine resistance, recombinant plasmids of wild type (3D7) and highly pyrimethamine resistant (7G8) gene were constructed by cassette mutagenesis using Nco1 -Xba1 fragments from the corresponding cloned TS-DHFR genes. Characterization of the purified-recombinant enzymes with regard to their kinetic properties and inhibition by pyrimethamine strongly clearly indicated that point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.
KeywordsDHFR Gene Ribosomal Binding Sequence Pyrimethamine Resistance DHFR Activity Report Nucleotide Sequence
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