Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.
Retroviral infection requires integration of the viral genome into the host genome. Recombinant integrase proteins may be purified following bacterial expression. A bulk biochemical assay of integrase function relies on the conversion of supercoiled plasmids to linear or relaxed circles. Single molecule molecular tweezer assays of integrase also evaluate the conversion of supercoiled DNA to nicked and broken species. A bacterial nuclease that co-purifies with retroviral integrase may affect the quantitation of integration activity in bulk or single molecule assays. During purification of retroviral integrase from bacteria, fractions may be screened for contaminating nuclease activity. In order to efficiently separate the nuclease from integrase, the binding affinities of each protein must differ. We find that a co-purifying nuclease may be efficiently separated from integrase based on heparin affinity, but not ionic affinity.
Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, United States.
Miguel A
MA
Lopez
Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, United States.
Randi M
RM
Mackler
Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, United States. Electronic address: yoder.176@osu.edu.
Kristine E
KE
Yoder
D002846:Chromatography, Affinity; D004279:DNA, Viral; D003851:Deoxyribonucleases; D006493:Heparin / Q000737:chemistry; D019426:Integrases / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry; D016092:Spumavirus / Q000201:enzymology* / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016662:Virus Integration
D004279:DNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D006493:Heparin; D012685:Sepharose; D019426:Integrases; D003851:Deoxyribonucleases
Integrase; Protein purification; Retrovirus