Preparation and characterization of a highly soluble Aβ_1-42 peptide variant

Alzheimer's disease (AD) is a progressive neurological disease marked by the accumulation and deposition of misfolded amyloid beta or Abeta (Aβ) peptide. Two species of Aβ peptides are found in amyloid plaques, Aβ_1-40 and Aβ_1-42, with the latter being the more amyloidogenic of the two. Understanding how and why Aβ peptides misfold, oligomerize and form amyloid plaques requires a detailed understanding of their structure and dynamics. The poor solubility and strong aggregation tendencies of Aβ_1-42 has made the isolation and characterization of its different structural isoforms (monomer, dimer, oligomer, amyloid) exceedingly difficult. Furthermore, while synthetic Aβ_1-42 peptides (Aβ₄₂syn) are readily available, the cost of isotopically labeled peptide is substantial, making their characterization by NMR spectroscopy cost prohibitive. Here we describe the design, cloning, high-level production, isotopic labeling and biophysical characterization of a modified (solubility-tagged) Aβ_1-42 variant that exhibits excellent water solubility and shares similar aggregation properties as wildtype Aβ_1-42. Specifically, we attached six lysines (6K) to the C-terminus of native Aβ_1-42 to create a more soluble, monomeric form of Aβ_1-42 called Aβ₄₂C6K. A gene for the Aβ₄₂C6K was designed, synthesized and cloned into Escherichia coli (E. coli) and the peptide was expressed at milligram levels. The Aβ₄₂C6K peptide was characterized using circular dichroism (CD), NMR, electron microscopy and thioflavin T fluorescence. Its ability to form stable monomers, oligomers and fibrils under different conditions was assessed. Our results indicate that Aβ₄₂C6K stays monomeric at high concentrations (unlike Aβ_1-42) and can be induced to oligomerize and form fibrils like Aβ_1-42. Our novel construct could be used to explore the structure and dynamics of Aβ_1-42 as well as the interaction of ligands with Aβ_1-42 via NMR.