Sammanfattning

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.

Nyckelord

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

Publicerad i

Biotechnology Journal
2015, Volym: 10, nummer: 11, sidor: 1707-1718
Utgivare: WILEY-V C H VERLAG GMBH

SLU författare

• Härd, Torleif

  • Institutionen för kemi och bioteknologi, Sveriges lantbruksuniversitet
    

UKÄ forskningsämne

Biokemi och molekylärbiologi
Cellbiologi
Botanik


Publikationens identifierare

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

Permanent länk till denna sida (URI)

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