title: A phosphorylation tag for uranyl mediated protein purification and photo assisted tag removal.
abstract: Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO22+) may provide a dual function in protein purification and subsequent C-terminal tag removal procedures.
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Niels Erik
authors.initials: NE
authors.lastname: Møllegaard
mesh_terms: D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008027:Light; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012685:Sepharose; D021241:Spectrometry, Mass, Electrospray Ionization; D014501:Uranium / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014501:Uranium; D012685:Sepharose; D047390:Casein Kinase II