Probing Alzheimer amyloid peptide aggregation using a cell-free fluorescent protein refolding method

Fibrillation of the Alzheimer β-amyloid peptide (Aβ) and (or) formation of toxic oligomers are key pathological events in Alzheimer’s disease. Several strategies have been introduced to identify small molecule aggregation inhibitors, and based on these methods, a number of aggregation inhibitors have been identified. However, most of these methods use chemically synthesized Aβ42 peptides, which are difficult to maintain in a monomeric state at neutral pH where anti-aggregation screening is usually carried out. We have developed a cell-free Aβ42 aggregation assay based on fluorescence protein refolding. This assay utilizes nanomolar concentrations of protein. We genetically fused Aβ42 to the N-terminus of vYFP, a variant of of GFP, and expressed and purified the folded fusion protein. The refolding efficiency of Aβ42–vYFP fusion was inversely correlated with the solubility of Aβ42. Using fluorescence to monitor refolding of Aβ42–vYFP, we confirmed that Zn2+ binds to Aβ42 and increases its aggregation. The IC50 value estimated for Zn binding is 3.03 ± 0.65 µmol/L. We also show that this technique is capable of monitoring the aggregation of chemically synthesized Aβ42.