fulltext.study @t Gmail

Selection and characterization of Affibody ligands binding to Alzheimer amyloid β peptides

Paper ID Volume ID Publish Year Pages File Format Full-Text
25427 43573 2007 22 PDF Available
Title
Selection and characterization of Affibody ligands binding to Alzheimer amyloid β peptides
Abstract

Affibody® (Affibody) ligands specific for human amyloid beta (Aβ) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Aβ peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Aβ peptides from human plasma and serum. It was found that both ligands could efficiently capture Aβ that were spiked (100 μg ml−1) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30–36 of Aβ, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Aβ (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Aβ peptide, which we speculate to be dimeric Aβ. In conclusion, we have successfully selected Affibody ligands that efficiently capture Aβ peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Aβ peptides in order to slow down or reduce amyloid plaque formation, is discussed.

Keywords
Affibody ligand; Amyloid beta peptide; Serum depletion; Phage display; Protein engineering
First Page Preview
Selection and characterization of Affibody ligands binding to Alzheimer amyloid β peptides
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 128, Issue 1, 30 January 2007, Pages 162–183
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us