title: A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.
abstract: 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 KD 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 Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Lindberg
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
authors.forename: Torleif
authors.initials: T
authors.lastname: Härd
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden. ssta@kth.se.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D061505:Cell Surface Display Techniques / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D008958:Models, Molecular; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013210:Staphylococcus / Q000737:chemistry / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins
keywords: Affibody molecules; Affinity maturation; Amyloid beta; Bacterial display; Combinatorial protein engineering