title: High-affinity binders selected from designed ankyrin repeat protein libraries.
abstract: 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 the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.
authors.affiliation: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: H Kaspar
authors.initials: HK
authors.lastname: Binz
authors.forename: Patrick
authors.initials: P
authors.lastname: Amstutz
authors.forename: Andreas
authors.initials: A
authors.lastname: Kohl
authors.forename: Michael T
authors.initials: MT
authors.lastname: Stumpp
authors.forename: Christophe
authors.initials: C
authors.lastname: Briand
authors.forename: Patrik
authors.initials: P
authors.lastname: Forrer
authors.forename: Markus G
authors.initials: MG
authors.lastname: Grütter
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D011487:Protein Conformation