title: Overexpression and purification of enzymatically active recombinant integrase protein of Rous sarcoma virus.
abstract: The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control of lac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl-beta-D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity in Escherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn2+ over Mg2+, and showed substrate specificity at 1 mM MnCl2.
authors.affiliation: Department of Biology, Albert Szent-Györgyi Medical University, Szeged, Hungary.
authors.forename: I
authors.initials: I
authors.lastname: Marczinovits
authors.forename: J
authors.initials: J
authors.lastname: Molnár
authors.forename: J
authors.initials: J
authors.lastname: Sóki
authors.forename: I
authors.initials: I
authors.lastname: Fodor
mesh_terms: D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004926:Escherichia coli; D019426:Integrases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004278:DNA, Superhelical; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases