High-yield production and characterization of biologically active GST tagged human topoisomerase IIalpha protein in insect cells for the development of a high-throughput assay.
Summary of "High-yield production and characterization of biologically active GST tagged human topoisomerase IIalpha protein in insect cells for the development of a high-throughput assay."
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 IIalpha 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 IIalpha 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 IIalpha 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 IIalpha 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.
Affiliation
Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW. U.K.
Journal Details
This article was published in the following journal.
Name: Protein expression and purification
ISSN: 1096-0279
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/20709174
- DOI: http://dx.doi.org/10.1016/j.pep.2010.08.001
Medical and Biotech [MESH] Definitions
Sermorelin
The biologically active fragment of human growth hormone-releasing factor, consisting of GHRH(1-29)-amide. This N-terminal sequence is identical in several mammalian species, such as human, pig, and cattle. It is used to diagnose or treat patients with GROWTH HORMONE deficiency.
Sequence Tagged Sites
Short tracts of DNA sequence that are used as landmarks in GENOME mapping. In most instances, 200 to 500 base pairs of sequence define a Sequence Tagged Site (STS) that is operationally unique in the human genome (i.e., can be specifically detected by the polymerase chain reaction in the presence of all other genomic sequences). The overwhelming advantage of STSs over mapping landmarks defined in other ways is that the means of testing for the presence of a particular STS can be completely described as information in a database.
Teriparatide
A polypeptide that consists of the 1-34 amino-acid fragment of human PARATHYROID HORMONE, the biologically active N-terminal region. The acetate form is given by intravenous infusion in the differential diagnosis of HYPOPARATHYROIDISM and PSEUDOHYPOPARATHYROIDISM. (Reynolds JEF(Ed): Martindale: The Extra Pharmacopoeia (electronic version). Micromedex, Inc, Englewood, CO, 1995)
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9,10-Secoergosta-5,7,10(19),22-tetraene-3,25-diol. Biologically active metabolite of vitamin D2 which is more active in curing rickets than its parent. The compound is believed to attach to the same receptor as vitamin D2 and 25-hydroxyvitamin D3.
Nanoconjugates
Tailored macromolecules harboring covalently-bound biologically active modules that target specific tissues and cells. The active modules or functional groups can include drugs, prodrugs, antibodies, and oligonucleotides, which can act synergistically and be multitargeting.
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