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 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.
Zhelkovsky
A M
AM
Department of Molecular Biology and Microbiology, School of Veterinary Medicine, Tufts University, Boston, Massachusetts 02111-1800.
Moore
C L
CL
D000818:Animals; D002478:Cells, Cultured; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology
D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I