Expression, purification and characterization of recombinant mitochondrial topoisomerase II of kinetoplastid Crithidia fasciculata in High-five insect cells.
Topoisomerase II of kinetoplastid parasites plays an important role in the replication of unusual networks of kinetoplast DNA (kDNA) and is a very useful target for antiparasitic drugs. In this study, we cloned full-length Crithidia fasciculata mitochondrial topoisomerase II gene into pFastBac-HTc vector and successfully expressed an active recombinant full-length mitochondrial topoisomerase II in Bac-to-Bac baculovirus expression system. A rapid and simple purification strategy was established by incorporating a FLAG-tag at the C-terminus of the protein. The purified recombinant topoisomerase II showed a major single band on SDS-PAGE (>96% purity) and was verified through Western blot analysis. The recombinant full-length mitochondrial topoisomerase II exhibited decatenation, catenation and relaxation activity of type II topoisomerase as well as various sensitivities to a series of known topoisomerase inhibitors. These studies explore new way and lay groundwork to express all other similar full-length kinetoplastid topoisomerases, it will also facilitate further elucidation of X-ray structure, catalysis mechanism of kinetoplastid topoisomerases and design of new antiparasitic drugs targeting kinetoplastid topoisomerases.
Wang
Yuzhen
Y
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Tsai
Yu-Cheng
YC
Urena-Rivera
Esteban
E
Chen
Junghuei
J
D000818:Animals; D002460:Cell Line; D016831:Crithidia fasciculata / Q000201:enzymology* / Q000235:genetics; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000378:metabolism*; D015870:Gene Expression; D008928:Mitochondria / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D018105:DNA, Kinetoplast; D011994:Recombinant Proteins; D004250:DNA Topoisomerases, Type II