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Research article - Peer-reviewed, 2015

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity

Lindberg, Hanna; Härd, Torleif; Löfblom, John; Ståhl, Stefan

Abstract

The amyloid hypothesis suggests that accumulation of amyloid (A) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited A aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the A peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the A peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity A-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds A with a K-D value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture A(1-42) peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the A peptide.

Keywords

Affibody molecules; Affinity maturation; Amyloid beta; Bacterial display; Combinatorial protein engineering

Published in

Biotechnology Journal
2015, Volume: 10, number: 11, pages: 1707-1718
Publisher: WILEY-V C H VERLAG GMBH

      SLU Authors

    • Härd, Torleif

      • The Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences

    UKÄ Subject classification

    Biochemistry and Molecular Biology
    Cell Biology
    Botany

    Publication identifier

    DOI: https://doi.org/10.1002/biot.201500131

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/73424