Abstract
BrkA is a type Vα autotransporter-based display systems from Bordetella pertussis whose passenger region can be engineered for recombinant protein display on the bacterial surface, where displayed proteins become extracellularly accessible for detection and ligand binding. E3 is an amyloid-β-targeting nanobody directed against a key pathological peptide in Alzheimer’s disease, and in this study we examined whether pJMMS, a plasmid encoding a BrkA-E3 fusion, retains a BrkA-like surface-accessibility pattern in Escherichia coli UT5600. Because anti-Aβ nanobodies may be useful in future surface-display-based screening and targeting strategies, it is important to determine whether E3 incorporation disrupts BrkA processing or accessibility. AlphaFold modelling suggested that the engineered fusion retained a largely BrkA-like architecture, although these models were used only to assess structural plausibility rather than as direct evidence of export or surface display. Anti-His Western blotting showed that pJMMS exhibited a trypsin-sensitive lower-molecular weight band similar to that of the positive BrkA control. Growth-curve analysis further suggested that expression of BrkA-E3 did not substantially impair bacterial growth relative to pENS. Together, these findings support the conclusion that E3 incorporation is compatible with BrkA processing and formation of a surface accessible BrkA-derived species.