Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I target for antitumor drugs

The gene encoding human DNA topoisomerase (topo) I, the target of numerous anticancer drugs, has been subcloned into bacterial, yeast and baculovirus- based expression systems in attempts to overexpress the enzyme for extensiv e structural and functional characterisation. Expression in E.coli produced a protein which was not suitable for structural studies. Expression in the yeast system was more successful enabling the enzyme to be purified and ch aracterised. However, the resulting yield was modest for our requirements a nd the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that top I from human placental ti ssue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fr agment and shown by N-terminal sequence analysis that it starts at Lysine-1 91. This information was used to construct vectors which direct the overexp ression of this fragment in baculovirus infected insect cells. The recombin ant protein has been purified to homogeneity in a yield of 5-10mg/l of cel culture. The fragment is stable and retains all of the DNA driving activiti es of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides a nd conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichro ism technique has been further developed and applied for real-time monitori ng of supercoiled (sc) DNA relaxation by the enzyme and for comparative ana lysis of inhibition of 68kDa topo I by camptothecin (CPT).