title: Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications.
abstract: A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Chevrel
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Agathe
authors.initials: A
authors.lastname: Urvoas
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Ines
authors.initials: I
authors.lastname: Li de la Sierra-Gallay
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Aumont-Nicaise
authors.affiliation: Translational Research Department, Institut Curie, CNRS UMR 144, 26, rue d'Ulm, F_75248, Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Moutel
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Desmadril
authors.affiliation: Translational Research Department, Institut Curie, CNRS UMR 144, 26, rue d'Ulm, F_75248, Paris, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Perez
authors.affiliation: Ecole Polytechnique CNRS UMR7654, 91128 Palaiseau Cedex, France.
authors.forename: Alexis
authors.initials: A
authors.lastname: Gautreau
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Herman
authors.initials: H
authors.lastname: van Tilbeurgh
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France philippe.minard@u-psud.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Minard
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Valerio-Lepiniec
mesh_terms: D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D049452:Green Fluorescent Proteins
keywords: combinatorial library; green fluorescent protein (GFP); protein design; protein interactions; repeat protein; αRep