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High-affinity binders selected from designed ankyrin repeat protein libraries


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 Å 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.

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Figure 1: Construction of designed AR protein libraries.
Figure 2: Expression, purification and SPR analysis of selected AR proteins.
Figure 3: ELISAs with selected AR proteins.
Figure 4: Crystal structure of the designed AR protein off7 in complex with MBP.
Figure 5: Open sandwich illustrations of the interaction surfaces of AR proteins and their targets.

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We thank the members of the Plückthun and Grütter laboratories for valuable discussions and David L. Zechel for the critical reading of the manuscript. H.K.B. was supported by a pre-doctoral fellowship of the Roche Research Foundation. M.T.S. was in receipt of a pre-doctoral scholarship from the Fonds der Chemischen Industrie and the Bundesministerium für Bildung und Forschung. This work was supported by the Swiss National Center of Competence in Research in structural biology and the Swiss Krebsliga.

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Correspondence to Markus G Grütter or Andreas Plückthun.

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The technology in this paper has been patented by M.T.S., P.F, H.K.B. and A.P.

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Binz, H., Amstutz, P., Kohl, A. et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 22, 575–582 (2004).

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