Abstract
Autotransporters are a class of auto-display proteins used extensively in bacterial surface display systems, where a passenger domain is translocated to the cell exterior via a β-barrel domain anchored in the outer membrane. The hemoglobin-binding protease (Hbp) β-barrel has been established as a favourable translocator for chimeric autotransporter systems due to its high efficiency of outer membrane integration; however, its capacity to translocate the BrkA passenger domain from Bordetella pertussis has not been previously determined. In this study, we investigated whether the Hbp β-barrel could functionally replace the native BrkA β-barrel to support the surface display of the BrkA passenger domain in Escherichia coli BL21(DE3) by engineering and evaluating the chimeric plasmid pHAD (plasmid Hbp AutoDisplay). Sequence verification revealed a frameshift mutation within the Hbp β-barrel C-terminal region. Despite this, cells harbouring pHAD showed a significant increase in growth rate, and reduced but detectable levels of BrkA passenger domain in the membrane fraction. These results are suggestive of membrane association and partial surface exposure of the BrkA passenger domain mediated by the Hbp β-barrel, while highlighting the need for construct correction and optimized detection conditions to fully assess the surface-display capacity of this chimeric system.