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- Title
The aRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications.
- Authors
Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Guellouz, Asma; Ferrandez, Yann; Mesneau, Agnès; Li de la Sierra-Gallay, Ines; Aumont-Nicaise, Magali; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe
- Abstract
We have designed a new family of artificial proteins, named aRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an a-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 aRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. aRep 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 aRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that aReps can be used as bait in pulldown experiments. aReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments.
- Subjects
SYNTHETIC proteins; ELONGATION factors (Biochemistry); PHOSPHOPROTEIN phosphatases; X-ray crystallography; GENE expression
- Publication
Biochemical Society Transactions, 2015, Vol 43, Issue 5, p819
- ISSN
0300-5127
- Publication type
Article
- DOI
10.1042/BST20150075