Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.
Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil. The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designed based on E. coli codon preference with unique restriction sites evenly placed throughout the coding sequence. The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain. Each of these domains contained numerous unique restriction sites to facilitate mutagenesis. The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence. The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E. coli about 10-fold more efficiently than from the wild-type sequence. The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study. The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria.
Prapunwattana
P
P
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Sirawaraporn
W
W
Yuthavong
Y
Y
Santi
D V
DV
D000818:Animals; D001482:Base Composition; D003062:Codon; D016054:DNA, Protozoan / Q000138:chemical synthesis; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan; D005813:Genes, Synthetic; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D003062:Codon; D016054:DNA, Protozoan; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase