Härd, Torleif
- The Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences
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
UKÄ Subject classification
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