Overexpression of Human DNA Topoisomerase I in Insect Cells Using a Baculovirus Vector

doi:10.1006/prep.1994.1053

Protein Expression and Purification

Volume 5, Issue 4, August 1994, Pages 364-370

https://doi.org/10.1006/prep.1994.1053 Get rights and content

Abstract

The 3645-bp human DNA topoisomerase I cDNA isolated by D′Arpa et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 2543-2547) was integrated into the Autographa californica multiple nuclear polyhedrosis virus genome. The recombinant protein was expressed by infecting the SF9 insect cell line with this baculovirus and resulted in a 100-fold overexpression of human DNA topoisomerase I compared to the level found in human cell lines. This 100-kDa recombinant protein has the same electrophoretic mobility as the human DNA topoisomerase I from HeLa cells and is recognized by topoisomerase I-specific monoclonal antibody. The recombinant DNA topoisomerase I was isolated and purified to homogeneity with a two-step fractionation protocol and has a specific activity of 2 × 10⁶ U/mg. Enzymatic properties such as stimulation by magnesium and inhibition by camptothecin resemble properties of the enzyme purified from human cell lines.

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Cited by (36)

RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription
2016, Cell
Citation Excerpt :
Human RNAPII and general transcription factors were purified according to Maldonado et al. (1996). Human recombinant TOP1 and BRD4 were purified as described, respectively in Zhelkovsky and Moore (1994) and Maruyama et al. (2002). TOP1 was incubated with equimolar amount of recombinant factors for 6 min in ice in TOP1 buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 ug/ml BSA).
We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.
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
2011, Protein Expression and Purification
Citation Excerpt :
Collectively, these data illustrate that the recombinant GST-htopo2α generated in our studies is a viable source of enzyme to use for inhibitor identification or selectivity studies. The insect cell expression system has previously been used to express human topoisomerase I [17] and recently was used in the expression of htopo2α [12] with a C-terminal PreScission-6× His Strep tag though their protein yields were not quoted in the paper. The introduction of a GST-tag at the N-terminus of htopo2α in our studies facilitated purification resulting in relatively high yields (40 mg/L) of active protein Thus we have set precedence for the high-yield expression and purification of GST-tagged htopo2α using the baculovirus insect cell system.
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/10⁹ 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. IC₅₀ 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.
Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies
2004, Bioorganic and Medicinal Chemistry
Human topoisomerase I (Top1) plays a pivotal role in cell replication and transcription, and therefore is an important anti-cancer target. Homocamptothecin is a lead compound for inhibiting Top1, and is composed of five conjugated planar rings (A–E). The homocamptothecin E-ring β-hydroxylactone opens slowly to a carboxylate at pH > 7.0. We analyzed, which form of homocamptothecin was biochemically relevant in the following ways: (1) the homocamptothecin carboxylate was tested for activity in vitro and found to be inactive; (2) homocamptothecin was incubated with Top1 and dsDNA, and we found that the homocamptothecin β-hydroxylactone form was stabilized; (3) the homocamptothecin E-ring β-hydroxylactone was modified to prevent opening, and the derivatives were either inactive or had low activity. These results indicated that the homocamptothecin β-hydroxylactone was the active form, and that an E-ring carbonyl oxygen and adjacent unsubstituted/unprotonated ring atom were required for full activity. Homocamptothecin and derivatives were docked into a Top1/DNA active site model, in which the +1 deoxyguanosine was rotated out of the helix, in order to compare the interaction energies between the ligands and the Top1/DNA active site with the in vitro activities of the ligands. It was found that the ligand interaction energies and in vitro activities were correlated, while the orientations of the ligands in the Top1/DNA active site explained the importance of the E-ring β-hydroxylactone independently of E-ring opening. An essential component of this Top1/DNA active site model is the rotated +1 deoxyguanosine, and in vitro experiments and molecular modeling studies supported rotation of the +1 deoxyguanosine out of the helix. These results allow for the rational design of more potent Top1 inhibitors through engineered interactions with as yet unutilized Top1 active-site residues including: Glu356, Asn430, and Lys751.
Topoisomerase I-DNA complexes contribute to arsenic trioxide-induced apoptosis
2004, Journal of Biological Chemistry
Citation Excerpt :
2-14C]Thymidine was obtained from PerkinElmer Life Sciences. Recombinant human Top1 was purified from TN5 insect cells (HighFive; Invitrogen) using a Baculovirus construct for the NH2-terminal truncated human Top1 cDNA as described previously (39, 40). Cell Culture—The human leukemic cell lines NB4 (APL-derived cells, kindly provided by Dr. M. Lanotte, INSERM U496, Hospital Saint-Louis, Paris, France) and U937 (American Type Culture Collection, Manassas, VA) and a U937-derivative cell clone containing the full-length bcl-2 cDNA (U937/Bcl-2, kindly provided by Dr J. Bréard, INSERM U461, Chatenay-Malabry, France) were grown in suspension in RPMI 1640 medium with glutamax-I (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Gemini Bio-Products, Woodland, CA) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin) in an atmosphere of 95% air and 5% CO2 at 37 °C.
Topoisomerase I is an essential enzyme that relaxes DNA supercoiling by forming covalent DNA cleavage complexes, which are normally transient. Topoisomerase I-DNA complexes can be trapped by anticancer drugs (camptothecins) as well as by endogenous and exogenous DNA lesions. We show here that arsenic trioxide (a potent inducer of apoptosis that induces the intracellular accumulation of reactive oxygen species and targets mitochondria) induces cellular topoisomerase I cleavage complexes. Bcl-2 overexpression and quenching of reactive oxygen species, which prevent arsenic trioxide-induced apoptosis, also prevent the formation of topoisomerase I-DNA complexes, whereas enhancement of reactive oxygen species accumulation promotes these complexes. The caspase inhibitor, benzyloxycarbonyl-VAD partially prevents arsenic trioxide-induced topoisomerase I-DNA complexes and apoptosis, suggesting that activated caspases further maintain intracellular levels of reactive oxygen species that induce the formation of topoisomerase I-DNA complexes. Down-regulation of topoisomerase I expression decreases arsenic trioxide-induced apoptotic DNA fragmentation. Thus, we propose that arsenic trioxide induces topoisomerase I-DNA complexes that participate in chromatin fragmentation and programmed cell death during apoptosis.
Isodiospyrin as a novel human DNA topoisomerase I inhibitor
2003, Biochemical Pharmacology
Isodiospyrin is a natural product from the plant Diospyros morrisiana, which consists of an asymmetrical 1,2-binaphthoquinone chromophore. Isodiospyrin exhibits cytotoxic activity to tumor cell lines but very little is known about its cellular target and mechanism of action. Unlike the prototypic human topoisomerase I (htopo I) poison camptothecin, isodiospyrin does not induce htopo I–DNA covalent complexes. However, isodiospyrin antagonizes camptothecin-induced, htopo I-mediated DNA cleavage. Binding analysis indicated that isodiospyrin binds htopo I but not DNA. These results suggest that isodiospyrin inhibits htopo I by direct binding to htopo I, which limits htopo I access to the DNA substrate. Furthermore, isodiospyrin exhibits strong inhibitory effect on the kinase activity of htopo I toward splicing factor 2/alternate splicing factor in the absence of DNA. Thus, these findings have important implications on naphthoquinone and its derivatives’ cellular mode of actions, i.e. these novel DNA topoisomerase I inhibitors can prevent both DNA relaxation and kinase activities of htopo I.
Interaction of human nuclear topoisomerase I with guanosine quartet-forming and guanosine-rich single-stranded DNA and RNA oligonucleotides
2002, Journal of Biological Chemistry
Citation Excerpt :
Briefly, the top1 gene was cloned into a baculovirus transfer vector (pBacGus-1, Novagen, Madison, WI) and used to make a recombinant baculovirus following the manufacturer's recommendations (BD-PharMingen, San Diego, CA). Top1 protein was then expressed in TN5 insect cells (HighFive, Invitrogen) by the recombinant baculovirus and purified via the N-terminal His tag essentially as described (26, 38). 3′-End-labeling was performed using terminal deoxynucleotidyl transferase (Invitrogen) with [α-32P]cordycepin 5′-triphosphate as described previously (39).
Human nuclear DNA topoisomerase I (top1) plays a crucial role in DNA replication, transcription, and chromosome condensation. In this study, we show that intra- and intermolecular guanosine quartets (G-quartets) can inhibit top1-mediated DNA cleavage at a high affinity site. Top1-mediated DNA cleavage was also inhibited by a 16-mer single-stranded oligodeoxynucleotide (ODN) containing a G-rich sequence (G₂T₂G₅TG₂TG₃) and by its RNA equivalent, neither of which form G-quartet structures. A comparison of various single-stranded ODN for their ability to inhibit top1-mediated DNA cleavage indicated that G-rich sequences containing repeats of 2 or 3 consecutive guanines interspaced with thymines specifically inhibited top1. We also found that both single-stranded and G-quartet-forming ODNs bind to top1 without being cleaved by the enzyme. These results demonstrate that either DNA or RNA G-rich single-stranded and G-quartet-forming oligonucleotides can bind to top1 and prevent cleavage of duplex DNA.

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Regular ArticleOverexpression of Human DNA Topoisomerase I in Insect Cells Using a Baculovirus Vector

Abstract

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Overexpression of Human DNA Topoisomerase I in Insect Cells Using a Baculovirus Vector