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Protein Expr Purif. 1994 Aug;5(4):364-70.

Overexpression of human DNA topoisomerase I in insect cells using a baculovirus vector.

Author information

1
Department of Molecular Biology and Microbiology, School of Veterinary Medicine, Tufts University, Boston, Massachusetts 02111-1800.

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 x 10(6) U/mg. Enzymatic properties such as stimulation by magnesium and inhibition by camptothecin resemble properties of the enzyme purified from human cell lines.

PMID:
7950383
DOI:
10.1006/prep.1994.1053
[Indexed for MEDLINE]

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