Materials and Methods

Cloning. Primers were designed based upon malarial genomic DNA sequences flanking genes of interest and incorporating restriction sites and a protease cleavable His tag sequence. Pyrimidine genes were transcribed by PCR from P. falciparum 3D7 genomic DNA with the extension step at 65°C rather than 72°C due to the high AT content. The PCR product was ligated into a pET expression vector. E. coli strain PMC103 was used as the primary transformant host as it has reduced recombination mechanisms (7).

Over-Expression and Purification. E. coli BL-21 (DE3) cells were transformed with the expression plasmid and the hybrid plasmid, pMICO, containing both pLysS expressing T7 lysozyme which inhibits RNA polymerase, and the RIG plasmid expressing rare tRNAs for Arg, Ile and Gly (8). Bacteria were induced with 0.5 mM IPTG, the temperature was reduced to 25°C, and cells were grown for a further 4–16 h. Cells were then lysed and the soluble fraction was applied to a Ni-NTA chromatography column, and the His tagged protein was purified using step-wise elution with imidazole buffers. Enzymes were further purified as required by ion-exchange chromatography on a Poros HQ cartridge using a Biocad Model 700E. Radioassays for DHOase, OPRTase and ODCase have been described elsewhere (4).