title: The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications.
abstract: We have designed a new family of artificial proteins, named αRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an α-helical repeated motif. The sequence of the repeated motifs, first identified in a thermostable archae protein was optimized using a consensus design strategy and used for the construction of a library of artificial proteins. All proteins from this library share the same general fold but differ both in the number of repeats and in five highly randomized amino acid positions within each repeat. The randomized side chains altogether provide a hypervariable surface on αRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. αRep binders with high affinity for various protein targets were selected by phage display. Low micromolar to nanomolar dissociation constants between partners were measured and the structures of several complexes (specific αRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that αReps can be used as bait in pull-down experiments. αReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments.
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
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: 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: Asma
authors.initials: A
authors.lastname: Guellouz
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: Yann
authors.initials: Y
authors.lastname: Ferrandez
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: Agnès
authors.initials: A
authors.lastname: Mesneau
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 Li
authors.initials: IL
authors.lastname: 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: 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: 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
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D003460:Crystallization / Q000379:methods*; D055785:Gene Knockdown Techniques; D015723:Gene Library; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins
keywords: combinatorial library; crystal structure; protein design; repeat protein; αRep