Topoisomerase II of the Malarial parasite

of Thesis. The gene encoding topoisomerase II of P. falciparum (PfTopoll) was isolated and characterised in 1994 (Cheesman et al.,) as part of an overall strategy to isolate parasite genes involved in DNA replication and evaluate their potential as anti-malarial drug targets. As a natural progression, this study has focussed on heterologous expression of the gene to enable sufficient recombinant protein to be purified for biochemical analysis and drug screening. The species divergent Cterminal region of the protein was expressed in E. coli, and used to raise polyclonal antiserum in rabbits, which detects a protein of 160/175 kDa in extracts from P. falciparum. The same antiserum was used to monitor expression of recombinant PfTopoll in baculovirus. A triplet of proteins of 160/175 kDa were detected by the antiserum in cell extracts derived from baculovirus-infected insect cells, but in very small quantities. This is close to the predicted size of PfTopoll. Incorporation of a C-terminal hexahistidine affinity tag into the protein was used as a means to purify the small amount of recombinant protein that was made, using nickel affinity chromatography. Decatenation assays, which are a specific test for type II topoisomerase activity, detected activity in some fractions obtained from the affinity purification. However, a mock purification, using an unrelated recombinant baculovirus gave similar results, indicating that a contaminating co-purifying topoisomerase II activity was present. Full length expression of recombinant PfTopoll was not detected in cell extracts prepared from E. coil. However, there is evidence that the recombinant protein was expressed, but may have undergone rapid proteolytic degradation in vivo. Expression of a functionally inactive version of the protein, in which the active site, tyrosine, was changed to phenylalanine, had no noticeable affect on protein stability. A modified version of the PfTopoll gene, in which the first four codons were replaced with the first five codons of the yeast topoisomerase II gene was expressed in S. cerevisiae. A control plasmid expressed the human topoisomerase II c homologue to high levels, but no evidence of expression of recombinant PfTopoll was detected. Northern analysis of the mRNA revealed the presence of truncated transcripts, and RT-PCR mapped the sites of polyadenylation to 370 and 376 nucleotides from the AUG. An analysis of the region upstream of these sites revealed the presence of specific AT-rich sequence elements which are known to cause transcription termination of yeast mRNAs. Site directed mutagenesis was used to change these sequences, and analysis of the mRNA from the modified construct showed that the original transcriptional block had been abolished, but was replaced by a new transcriptional block, which gave rise to a truncated transcript some 617 nt in length. An investigation into the developmental regulation of PfTopoll throughout the blood stages of parasite growth was also undertaken. Northern analysis indicates that three transcripts accumulate principally in the trophozoite stage, although interestingly, work conducted by Paul Horrocks indicates that the promoter is active in both ring and trophozoite/schizont stage parasites. Western analysis of protein extracts derived from synchronised populations of parasites shows that trophozoite and schizont stages express a triplet of proteins of 160/175 kDa in size, and at similar levels. Although protein extracts from trophozoite and schizont stage parasites are proficient at decatenation, activity was highest in schizonts, and could be inhibited completely after immunodepleting the parasite extracts.