Cloning and expression of topoisomerase genes from Trypanosoma brucei

Topoisomerases (TOPs) are essential enzymes that catalysis topological changes in DNA. TOPs are pivotal to cell survival and therefore inhibitors have been developed to target these enzymes both for antimicrobial and anti-cancer chemotherapy. The genome of the protozoan parasite Trypanosoma brucei contains 7 different genes for TOPs (TbTOPIA, TbTOPIB, TbTOPIImt, TbTOPIIα, TbTOPIIβ, TbTOPIIIα and TbTOPIIIβ). By using RNAi, it was shown that TbTOPIA, TbTOPIB, TbTOPIImt and TbTOPIIα are essential for parasite growth. These findings indicate that these four TbTOPs are good targets for anti-trypanosomal drug development. The genetic validations of the TbTOPs as suitable anti-trypanosomal drug targets are corroborated by inhibitor studies. It has been shown that different TOP inhibitors, including approved anti-cancer drugs, exhibit promising trypanocidal activities. In order to carry out screening assays to identify TOP inhibitors as new chemotherapies against T. brucei, it is necessary to produce considerable amounts (mg quantities) of active TbTOPs.

Objectives of the PhD
The aim of the project is to recombinantly expressed TbTOPs and to purify the enzymes in active form suitable for inhibitor screening assays. The individual measurable objectives are:
(i) to clone and express TbTOPs as His-tagged proteins,
(ii) to purify His-tagged TbTOPs and to determine their enzymatic activities, and
(iii) to screen compound libraries for specific inhibitors of TbTOPs.
The gene sequences of all TbTOPs are available, and these will be utilized to construct primers for the cloning of these genes in His expression vectors. His-tagged TbTOPs will be purified from cell extracts using Ni2+-NTA agarose columns. The activity of the purified TbTOPs will be determined by using topoisomerase assay kits. Recombinant TbTOPs will then be used for screening small compound libraries using a recently developed high-throughput assay. Promising compounds will be tested for their trypanocidal activity in cell culture with bloodstream forms of T. brucei.