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A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity
Author(s) -
Lindberg Hanna,
Härd Torleif,
Löfblom John,
Ståhl Stefan
Publication year - 2015
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201500131
Subject(s) - isolation (microbiology) , chemistry , bacteria , beta (programming language) , biophysics , amyloid (mycology) , computational biology , biochemistry , combinatorial chemistry , microbiology and biotechnology , nanotechnology , biology , computer science , genetics , materials science , inorganic chemistry , programming language
The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ 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 Aβ 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 Aβ 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 Aβ‐binding molecules were isolated using flow‐cytometric sorting. The best performing candidate binds Aβ 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 Aβ 1‐42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.