Abstract
DNA topoisomerase I (topo I) can be isolated from cultured human cells in quantities that are more than sufficient for investigations into the ability of topo I to relax supercoiled DNA (250 μg/109 cells) (1,2). However, the production of human topo I (htopo I) in this manner becomes both costly and laborintensive if milligram quantities are needed for structural studies. Although active htopo I has been overexpressed in mammalian cells (3), yeast (4), and Escherichia coli (5,6), these systems have not proven capable of providing large quantities of the protein. In E. coli, the htopo I gene was found to be highly toxic to most strains (3) and appears to contain cryptic prokaryotic promoter elements that lead to constitutive expression of truncated forms of the protein (Madden and Champoux, unpublished observations). In addition, the E. coli-expressed htopo I is very unstable, with proteolytic breakdown products nearly as abundant as those of the full-length protein (6). This is true for htopo I constructs fused to either the T7 gene 10 translation initiation signal (6) or to glutathione-S-transferase (GST). Furthermore, expression in protease-deficient strains of E. coli does not appreciably reduce the proteolytic breakdown of htopo I (unpublished observations).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Liu, L. F. and Miller, K. G. (1981) Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc. Natl. Acad. Sci. USA 78, 3487–3491.
Stewart, L., Ireton, G. C., Parker, L. H., Madden, K. R., and Champoux, J. J. (1996) Biochemical and biophysical analyses of recombinant forms of human topoisomerase I. J. Biol. Chem. 271, 7593–7601.
Madden, K. R. and Champoux, J. J. (1992) Overexpression of human topoisomerase I in baby hamster kidney cells: hypersensitivity of clonal isolates to camptothecin. Cancer Res. 52, 525–532.
Bjornsti, M. A., Benedetti, P., Viglianti, G. A., and Wang, J. C. (1989) Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the antitumor drug camptothecin. Cancer Res. 49, 6318–6323.
D’Arpa, P., Machlin, P. S., Ratrie, H. D., Rothfield, N. F., Cleveland, D. W., and Earnshaw, W. C. (1988) cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc. Natl. Acad. Sci. USA 85, 2543–2547.
Kikuchi, A. and Miyaike, M. (1993) Expression of human type I and II DNA topoisomerases and their derivatives in Escerichia coli, in Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy (Andoh, T., Ikeda, H., and Oguro, M., eds.), CRC, Boca Raton, FL, pp. 121–130.
Luckow, V. A. (1993) Baculovirus systems for the expression of human gene products. Curr. Opin. Biotechnol. 4, 564–572.
Stewart, L., Ireton, G. C., and Champoux, J. J. (1996) The domain organization of human topoisomerase I. J. Biol. Chem. 271, 7602–7608.
Stewart, L., Ireton, G. C., and Champoux, J. J. (1996) Reconstitution of human DNA topoisomerase I activity by fragment complementation. EMBO J. (Submitted.)
Zhelkovsky, A. M. and Moore, C. L. (1994) Overexpression of human DNA topoisomerase I in insect cells using a baculovirus vector. Protein Expr. Purif. 5, 364–370.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Holden, J. A., Rolfson, D. H., and Low, R. L. (1990) DNA topoisomerase I from human placenta. Biochim. Biophys. Acta. 1049, 303–310.
Caron, P. R. and Wang, J. C. (1994) Alignment of primary sequences of DNA topoisomerases, in DNA Topoisomerases and Their Applications in Pharmacology Advances in Pharmacology (Liu, L. F., ed.), Academic, Boca Raton, FL, pp. 271–291.
Alsner, J., Svejstrup, J. Q., Kjeldsen, E., Sorensen, B. S., and Westergaard, O. (1992) Identification of an N-terminal domain of eukaryotic DNA topoisomerase I dispensable for catalytic activity but essential for in vivo function. J. Biol. Chem. 267, 12,408–12,411.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
Stewart, L., Champoux, J.J. (1999). Purification of Baculovirus-Expressed Human DNA Topoisomerase I. In: Bjornsti, MA., Osheroff, N. (eds) DNA Topoisomerase Protocols. Methods in Molecular Biology, vol 94. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-259-7:223
Download citation
DOI: https://doi.org/10.1385/1-59259-259-7:223
Published:
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-0-89603-444-0
Online ISBN: 978-1-59259-259-3
eBook Packages: Springer Protocols