title: Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I-target for antitumor drugs.
abstract: 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 extensive 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 characterised. However, the resulting yield was modest for our requirements and the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that topo I from human placental tissue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fragment and shown by N-terminal sequence analysis that it starts at Lysine-191. This information was used to construct vectors which direct the overexpression of this fragment in baculovirus infected insect cells. The recombinant protein has been purified to homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and retains all of the DNA driving activities of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides and conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichroism technique has been further developed and applied for real-time monitoring of supercoiled (sc) DNA relaxation by the enzyme and for comparative analysis of inhibition of 68kDa topo I by camptothecin (CPT).
authors.affiliation: Department of Chemistry, University of York, United Kingdom. igor@yorvic.york.ac.uk
authors.forename: I B
authors.initials: IB
authors.lastname: Bronstein
authors.forename: A
authors.initials: A
authors.lastname: Wynne-Jones
authors.forename: A
authors.initials: A
authors.lastname: Sukhanova
authors.forename: F
authors.initials: F
authors.lastname: Fleury
authors.forename: A
authors.initials: A
authors.lastname: Ianoul
authors.forename: J A
authors.initials: JA
authors.lastname: Holden
authors.forename: A J
authors.initials: AJ
authors.lastname: Alix
authors.forename: G G
authors.initials: GG
authors.lastname: Dodson
authors.forename: J C
authors.initials: JC
authors.lastname: Jardillier
authors.forename: I
authors.initials: I
authors.lastname: Nabiev
authors.forename: A J
authors.initials: AJ
authors.lastname: Wilkinson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis* / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018411:Spodoptera; D059004:Topoisomerase I Inhibitors
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I