||Human DNA Topoisomerase I is the best studied of the DNA topoisomerase family. It catalyzes the relaxation of both positive and negative supercoils without the requirement of energy. In addition to DNA replication and transcriptional activation, DNA Topoisomerase I also plays a major role in pre-mRNA splicing, cell cycle, and other gene regulatory pathways during cell growth and development. The N-terminal 214 amino acids of Topoisomerase I comprise a highly charged N-terminal domain (NTD) involved in protein-protein interactions with a number of cellular proteins, including SV40 T antigen, nucleolin, SR proteins, p53 and other transcription factors. The N-terminal domain (NTD) of human DNA Topoisomerase I protein (residue 1-197) was expressed in baculovirus system and purified by using an affinity column and FPLC chromatography. Purified Topo I NTD can be tested forin vitrotranscription, pre-mRNA splicing, DNA binding and protein-protein interaction assays. Purified Topo I protein (NTD) is greater than 95% homogeneous and contains no detectable protease, DNase, and RNase activity.