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P2‐335: Supplementing α‐secretase cleavage of beta‐amyloid by targeted proteolytic antibody fragments using yeast display technology
Author(s) -
Kasturirangan Srinath,
Sierks Mike
Publication year - 2008
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2008.05.1412
Subject(s) - biotinylation , streptavidin , immunoglobulin light chain , chemistry , microbiology and biotechnology , peptide library , recombinant dna , biochemistry , antibody , amyloid (mycology) , phage display , amyloid beta , alpha secretase , yeast , peptide , peptide sequence , amyloid precursor protein , biology , biotin , gene , alzheimer's disease , genetics , medicine , inorganic chemistry , disease , pathology
Background: Deposition of beta-amyloid (A ) is considered an important early event in the pathogenesis of Alzheimer’s Disease (AD), and clearance of A represents a potential therapeutic approach. The recombinant antibody light chain fragment, mK18 was identified to have -secretase-like activity, producing the 1-16 and 17-40 amino acid fragments of A 40 as a primary product. In order to improve the targeting specificity of the proteolytic antibody for A , we have constructed a second generation library from a parent single chain antibody fragment (scFv) containing the variable domain of the proteolytic light chain and a random heavy chain. Methods: The scFv library was created by introducing random mutations in the CDR3 region of the heavy chain and subsequently expressing the library on the surface of yeast. Biotinylated covalently reactive analogs based on the -secretase site sequence were synthesized, immobilized on streptavidin beads, and used to select scFvs with increased specificity for A using magnetic bead enrichment. Collected cells were individually screened for proteolytic activity using 96-well plates and an internally quenched fluorogenic substrate that contains the -secretase site of A . Several individual clones with increased proteolytic activity were identified from 750 screened clones. Kinetic parameters were determined and compared. In-vitro studies using Thioflavin-T fluorescence, cytotoxicity using SHSY5Y cells and AFM imaging will be used to determine the effects of the second generation scFv on beta amyloid aggregation. Results: Second generation yeast display library was constructed in yeast. Two clones with highest binding affinity compared to the original clone were identified. Analysis of kinetic parameters (kcat and KM) of purified soluble scFv showed one clone had a 10-fold increase in activity (kcat/KM) toward the synthetic A substrate compared to the original scFv. Conclusions: Results on construction of the library, synthesis of analogs, the panning protocol and catalytic specificity and activity of selected clones will be presented. Catalytic activity toward A oligomers and fibrils and subsequent effects on aggregation and toxicity will be discussed.