Abstract
More than a century after its first identification, Alzheimer’s disease (AD) remains a neurodegenerative disorder with no cure. The accumulation of extracellular amyloid-beta (Aβ) plaques is a central pathological feature of AD, and impaired clearance contributes to disease progression. Previous in vitro studies have shown that physically coupling Aβ-targeting antibodies to Aβ-degrading enzymes, such as neprilysin (NEP), enhances the efficiency and specificity of amyloid cleavage by localizing enzymatic activity to the substrate. These findings motivate the development of co-expression-based clearance strategies in which targeting and degradation components function in close proximity. As an initial proof-of-concept toward a targeted co-expression clearance system, this study establishes a bacterial surface-display model to validate presentation of an Aβ-targeting nanobody in a controlled and accessible manner. Specifically, Escherichia coli was engineered to surface display the Aβ-binding nanobody E3 using the BrkA type V autotransporter system from Bordetella pertussis. A recombinant BrkA-based expression construct (pJMMS) was generated via Gibson Assembly® by fusing E3 to the 5′ end of the brkA passenger domain within the pENS backbone and transformed into E. coli UT5600. Colony PCR and nanopore sequencing confirmed correct insertion of the E3 sequence, and surface localization was verified using a trypsin accessibility assay. Together, these results establish a modular proof-of-concept platform for nanobody-guided targeting that can be extended to future co-expression of Aβ-degrading enzymes. This work provides a foundational engineering step toward targeted amyloid clearance systems, including future applications in engineered microglia.