title: High-yield production and characterization of biologically active GST-tagged human topoisomerase IIα protein in insect cells for the development of a high-throughput assay.
abstract: DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.
authors: Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Harlow, Essex, UK. praveen.singh@kcl.ac.uk
authors: Praveen K
authors: PK
authors: Singh
authors: Pan F
authors: PF
authors: Chan
authors: Martin J
authors: MJ
authors: Hibbs
authors: Maria-Jesus
authors: MJ
authors: Vazquez
authors: Delfina C
authors: DC
authors: Segura
authors: Daniel A
authors: DA
authors: Thomas
authors: Andrew J
authors: AJ
authors: Theobald
authors: Kathleen T
authors: KT
authors: Gallagher
authors: Namir J
authors: NJ
authors: Hassan
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D005982:Glutathione Transferase; D004250:DNA Topoisomerases, Type II