High-affinity binders selected from designed ankyrin repeat protein libraries

  title={High-affinity binders selected from designed ankyrin repeat protein libraries},
  author={H. Kaspar Binz and Patrick Amstutz and Andreas Kohl and Michael T. Stumpp and Christophe Briand and Patrik Forrer and Markus G. Grütter and Andreas Pl{\"u}ckthun},
  journal={Nature Biotechnology},
We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in… 
Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries.
A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm.
Selection of high-affinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions.
The generation of a library built upon the framework of a consensus FN3 domain sequence resulting in binding proteins the authors call Centyrins is described, which provides important data contributing to the understanding of potential FN3 binding interfaces and a new tool for generating high-affinity scaffold molecules.
Intracellular Kinase Inhibitors Selected from Combinatorial Libraries of Designed Ankyrin Repeat Proteins*
The use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors that bind APH with high specificity and with affinities down to the subnanomolar range is demonstrated.
Design, construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity
This work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.
Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)
The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well as the variable N-cap residues.
Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin
Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex.


Designed to be stable: Crystal structure of a consensus ankyrin repeat protein
The AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues, and represents an excellent basis for the design of novel binding molecules.
Structural basis for recognition by an in vitro evolved affibody
The crystal structure of a complex of an all α-helical in vitro selected binding protein (affibody) bound to protein Z, an IgG Fc-binding domain derived from staphylococcal protein A reveals an extended and complementary binding surface with similar properties to protein–antibody interactions.
An affibody in complex with a target protein: Structure and coupled folding
Comparisons in how the surface of the Z domain responds by induced fit to binding of ZSPA-1 and Ig Fc are noted, suggesting that the ZSPA-1 affibody is capable of mimicking the morphology of the natural binding partner for the Zdomain.
Engineered protein scaffolds for molecular recognition
  • A. Skerra
  • Biology, Chemistry
    Journal of molecular recognition : JMR
  • 2000
This review will concentrate on the critical description of the structural properties of experimentally tested protein scaffolds and of the novel functions that have been achieved on their basis, rather than on the methodology of how to best select a particular mutant with a certain activity.
Consensus-derived structural determinants of the ankyrin repeat motif
These generic Ankyrin repeat proteins can serve as prototypes for dissecting the rules of molecular recognition mediated by ankyrin repeats and for engineering proteins with novel biological functions, and suggest that statistical analysis and the consensus sequence approach can be used as an effective method to design proteins with complex topologies.
Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display
This work has mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.
Tailoring in vitro evolution for protein affinity or stability.
By a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible.