Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.
DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.
Lerner
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Pharmaceutical Products Research Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois, 60064-3500, USA.
Saiki
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D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001710:Biotin / Q000031:analogs & derivatives / Q000096:biosynthesis*; D002166:Camptothecin / Q000737:chemistry; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012588:Scintillation Counting; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D019809:Streptavidin; D014316:Tritium
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014316:Tritium; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin