Streamlined E. coli expression and purification of amyloid beta peptides via inclusion body formation and fluorescence monitoring

The recombinant production of amyloid beta peptides poses significant challenges due to their high aggregation propensity and cytotoxicity in bacterial hosts. In this study, we present a streamlined and reproducible method for the expression and purification of methionine-modified Aβ40 and Aβ42 peptides in Escherichia coli BL21 (DE3) pLysS. By leveraging inclusion body formation, the protocol enhances yield while simplifying purification. A key feature of this approach is the incorporation of real-time fluorescence spectroscopy and microscopy using Thioflavin-S and propidium iodide, enabling non-invasive monitoring of IB formation and expression dynamics. Purification was achieved through pH modulation, anion exchange chromatography, and ultrafiltration, yielding average peptide concentrations of 3.2 ± 1.3 mg/L for Aβ40 and 4.8 ± 3.2 mg/L for Aβ42. High-performance liquid chromatography confirmed average purities of 90.2 % ± 0.8 % for Aβ40 and 84.0 % ± 17.4 % for Aβ42. The structural integrity, aggregation kinetics, and neurotoxicity of the peptides were validated by PICUP, Thioflavin-T fluorescence assays, and cytotoxicity tests in primary hippocampal neurons. This tag-free, cost-effective platform provides a scalable solution for producing biologically active amyloid beta peptides, facilitating their use in structural biology, neurodegenerative disease research, and high-throughput drug screening.