title: Removal of impurities from transcription factor preparations that alter their DNA-binding properties.
abstract: Biochemical studies of transcriptional activators are important for understanding their detailed mechanism of action. Such experiments generally employ chimeric constructs comprised of fused DNA- binding and activation domains that are expressed in, and purified from, Escherichia coli, since full-length activators are usually difficult to express. We report here that such preparations contain chaperone impurities that affect the DNA-binding properties of the activator, for example sharply reducing the half-life of the protein-DNA complex. A simple method to remove these troublesome contaminants is described.
authors.affiliation: Department of Internal Medicine, University of Texas-Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8573, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Sun
authors.forename: Thomas
authors.initials: T
authors.lastname: Kodadek
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D016477:Artifacts; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006207:Half-Life; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C070633:Gal-VP16; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D004247:DNA