EXPRESSION AND PURIFICATION OF AMYLOID-BETA PEPTIDE 1-40: AN IN-DEPTH ANALYSIS FOR ISOLATION OF ITS MONOMERIC STATE

Amyloid-beta peptide 1-40 (Aβ40) is one of the main components involved in neurodegenerative diseases, and the accumulation of Aβ40 aggregates in the brain is considered a major contributing factor to Alzheimer’s disease. However, recent analyses have indicated that most Aβ40 on studies have not demonstrated its isolation in a monomeric state, which is a crucial starting point for understanding its pathological behavior. Therefore, this project aimed to develop an effective protocol to express and purify Aβ40, ensuring its isolation as a monomer. For that, Aβ40 was produced in inclusion bodies using recombinant protein expression in Escherichia coli. Aβ40 was purified from cellular impurities and solubilized in presence of a chaotropic agent. The isolation of Aβ40 in its monomeric state was ensured using Amicon system, followed by SEC-HPLC analysis. Then, SDS-PAGE technique was applied to verify the results obtained in previous steps, as well as dot blot assay to confirm Aβ40’s identity. Finally, aggregation kinetics were performed to confirm that Aβ40 was capable of aggregating under different conditions. Furthermore, this methodology was applied to produce isotopically labeled Aβ40 with ¹³C and ¹⁵N. The results demonstrated that the expression and purification steps were well established, as a significant amount of purified monomeric Aβ40 was achieved, both isotopically unlabeled and labeled, with their labels confirmed by mass spectrometry. The aggregation potential of Aβ40 was further demonstrated. Moving forward, the project will aim to analyze the interactions established in produced samples using NMR spectroscopy to understand the behavior of Aβ40 in its pathological state.