title: Purification of recombinant Rous sarcoma virus integrase possessing physical and catalytic properties similar to virion-derived integrase.
abstract: Recombinant Rous sarcoma virus integrase cloned from the Prague A (PrA) virus strain was expressed in Escherichia coli. Here we report the detailed purification procedure resulting in an apparently homogeneous integrase. Recombinant PrA integrase was compared at both the protein structural and the catalytic levels to avian myeloblastosis virus integrase purified from virions. Both proteins exist minimally in a dimeric state at low nanomolar concentrations as analyzed by glycerol gradient sedimentation and protein crosslinking studies. Likewise, both proteins have similar specific activities for full-site (concerted integration reaction) and half-site strand transfer activities using linear 480-bp retrovirus-like donor substrates that contain wild-type or mutant termini. They respond similarly to high NaCl concentrations ( approximately 350 mM) as well as aprotic solvents for efficient full-site strand transfer. The data suggest that recombinant integrase proteins with physical and catalytic properties similar to the virion counterpart can be purified using these techniques and will faithfully and efficiently promote the full-site integration reaction in vitro.
authors.affiliation: Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri, 63110, USA.
authors.forename: M
authors.initials: M
authors.lastname: McCord
authors.forename: S J
authors.initials: SJ
authors.lastname: Stahl
authors.forename: T C
authors.initials: TC
authors.lastname: Mueser
authors.forename: C C
authors.initials: CC
authors.lastname: Hyde
authors.forename: A C
authors.initials: AC
authors.lastname: Vora
authors.forename: D P
authors.initials: DP
authors.lastname: Grandgenett
mesh_terms: D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D019426:Integrases / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D014764:Viral Proteins; D019426:Integrases