A new method for the production of recombinant human Aβ(1-42) peptide

The amyloid-β protein (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Chemically synthesized Aβ(1-42) is the most widely employed resource for AD mechanistic research and drug screening. However, solid-phase synthesis introduces truncated and modified byproducts, causing batch-to-batch heterogeneity and compromised experimental reproducibility.

Herein, we established an optimized recombinant expression system utilizing a thermal green protein (TGP) fusion tag combined with TEV protease site-specific cleavage to produce tag-free, authentic human Aβ(1-42) in Escherichia coli. We systematically performed side-by-side biophysical characterization, including secondary structural transition and amyloid aggregation kinetics, to compare purified recombinant Aβ(1-42) (RecAβ) and conventional synthetic Aβ(1-42) (SynAβ). Human iPSC-derived neurons (iNs) and primary murine microglia were further applied to evaluate and compare their neurotoxicity and microglial regulatory functions.

The optimized TGP-TEV platform enabled robust production of high-purity RecAβ(1-42), yielding 6-7 mg intact peptide per liter of bacterial culture. Biophysical assays demonstrated that RecAβ shares highly conserved secondary structural features with SynAβ but exhibits significantly enhanced aggregation propensity. Functional assays revealed that RecAβ- and SynAβ-derived Aβ-derived diffusible ligands (ADDLs) exert equivalent neurotoxicity in human iNs. While both fibril preparations display comparable microglial binding recognition and lysosomal clearance kinetics with subtle temporal differences, RecAβ fibrils induce more severe microglial phagolysosomal dysfunction than SynAβ fibrils.

This work establishes a robust, reproducible recombinant strategy for generating authentic human Aβ(1-42). RecAβ recapitulates core AD-relevant bioactivities of commercial SynAβ while possessing stronger aggregation potency and enhanced capacity to disrupt microglial homeostatic function. This standardized recombinant Aβ preparation provides a useful tool for the investigation of Aβ biology and screening of AD therapeutic candidates.