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.
Highlights
Protein–protein interactions are essential to most biological functions in the cell
The three bGFPs and a non-relevant αRep control served as model proteins to compare
GFP binders selection The αRep library contains a repertoire of artificial repeat proteins from which specific binding molecules can be selected [26]
Summary
Protein–protein interactions are essential to most biological functions in the cell. Information concerning localization, trafficking, activation states and interaction partners of proteins in living cells is crucial to understand the complexity of cellular networks. Engineered recombinant antibody fragments of reduced size, such as single-chain variable fragment (ScFvs) are usually more efficiently produced. Some of these appear to fold in vivo and are compatible with intracellular applications. Single domain antibodies from camelidae (variable domain of heavy chain antibody (VHH) called nanobodies) or shark-derived antibody fragments [8], are more soluble and efficiently expressed c 2015 Authors.
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