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

Protein Expr Purif. 1994 Aug;5(4):364-70. doi: 10.1006/prep.1994.1053.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cells, Cultured
  • DNA Topoisomerases, Type I / biosynthesis*
  • DNA Topoisomerases, Type I / genetics
  • DNA Topoisomerases, Type I / isolation & purification
  • Genetic Vectors / genetics
  • Humans
  • Immunoblotting
  • Nucleopolyhedroviruses / genetics
  • Recombinant Proteins / biosynthesis
  • Spodoptera / cytology


  • Recombinant Proteins
  • DNA Topoisomerases, Type I