title: Purification of bacteriophage lambda repressor.
abstract: Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy "footprinting" assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.
authors.affiliation: Physics Department, Emory University, 400 Dowman Dr., Atlanta, GA 30322, United States.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Keith
authors.initials: K
authors.lastname: Shearwin
authors.forename: John
authors.initials: J
authors.lastname: Mack
authors.forename: Laura
authors.initials: L
authors.lastname: Finzi
authors.forename: David
authors.initials: D
authors.lastname: Dunlap
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D010582:Bacteriophage lambda / Q000235:genetics*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D000645:Ammonium Sulfate
keywords: Ammonium sulfate; Anion exchange; Bacteriophage lambda; Heparin affinity; His-tag; Lambda repressor; Single particle tracking; Tethered particle motion