CHEMICAL SYNTHESIS OF THE PLASMODIUM-FALCIPARUM DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE GENE

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DH FR-TS) is a well-known target for pyrimethamine and cycloguanil. The l ow amounts of enzyme obtainable from parasites or the currently availa ble heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designe d based on E. coli codon preference with unique restriction sites even ly placed throughout the coding sequence. The gene was designed and sy nthesized 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 syntheic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for fu rther study. The unique restriction sites in the coding sequence permi ts easy mutagenesis of the gene which should facilitate further unders tanding of the molecular basis of antifolate resistance in malaria. Co pyright (C) 1996 Elsevier Science B.V.