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.
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October 2015
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Review Article|
October 09 2015
The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications
Marie Valerio-Lepiniec;
Marie Valerio-Lepiniec
1
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Agathe Urvoas;
Agathe Urvoas
1
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Anne Chevrel;
Anne Chevrel
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Asma Guellouz;
Asma Guellouz
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Yann Ferrandez;
Yann Ferrandez
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Agnès Mesneau;
Agnès Mesneau
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Ines Li de la Sierra-Gallay;
Ines Li de la Sierra-Gallay
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Magali Aumont-Nicaise;
Magali Aumont-Nicaise
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Michel Desmadril;
Michel Desmadril
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Herman van Tilbeurgh;
Herman van Tilbeurgh
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
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Philippe Minard
Philippe Minard
2
*Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris-Sud, Bât 430, 91405 Orsay Cedex, France
2To whom correspondence should be addressed (emailphilippe.minard@u-psud.fr).
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Publisher: Portland Press Ltd
Received:
April 15 2015
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2015 Authors; published by Portland Press Limited
2015
Biochem Soc Trans (2015) 43 (5): 819–824.
Article history
Received:
April 15 2015
Citation
Marie Valerio-Lepiniec, Agathe Urvoas, Anne Chevrel, Asma Guellouz, Yann Ferrandez, Agnès Mesneau, Ines Li de la Sierra-Gallay, Magali Aumont-Nicaise, Michel Desmadril, Herman van Tilbeurgh, Philippe Minard; The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications. Biochem Soc Trans 1 October 2015; 43 (5): 819–824. doi: https://doi.org/10.1042/BST20150075
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