OVEREXPRESSION OF HUMAN DNA TOPOISOMERASE-I IN INSECT CELLS USING A BACULOVIRUS VECTOR

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 th e Autographa californica multiple nuclear polyhedrosis virus genome. T he recombinant protein was expressed by infecting the SF9 insect cell line with this baculovirus and resulted in a 100-fold overexpression o f human DNA topoisomerase I compared to the level found in human cell lines. This 100-kDa recombinant protein has the same electrophoretic m obility as the human DNA topoisomerase I from HeLa cells and is recogn ized by topoisomerase I-specific monoclonal antibody. The recombinant DNA topoisomerase I was isolated and purified to homogeneity with a tw o-step fractionation protocol and has a specific activity of 2 x 106 U /mg. Enzymatic properties such as stimulation by magnesium and inhibit ion by camptothecin resemble properties of the enzyme purified from hu man cell lines. (C) 1994 Academic Press, Inc.