Abstract
Breast cancer is the most frequently diagnosed cancer in women, and amplification of HER2 (human epidermal growth factor receptor 2), a tumour-associated antigen, defines an aggressive subtype while also providing a potential molecular marker for targeted therapies. This study investigates whether the Bordetella pertussis BrkA autotransporter system, which tolerates foreign protein insertion in its passenger domain, can be used to surface-display the HER2-specific nanobody C7b in Escherichia coli cells for targeted drug delivery and imaging applications. A recombinant BrkA-C7b plasmid (pBNJ) was constructed by inserting a C7b gene block into the 6x Histidine-tagged BrkA passenger domain within the pENS plasmid and expressed in UT5600 E. coli cells. Trypsin accessibility assays with western blotting confirmed the BrkA-C7b product to be surface accessible, however, trypsin susceptibility assays with western blotting indicated incomplete folding or misfolding of the construct, thereby suggesting a system that requires further optimization. These findings demonstrate surface display of C7b via the BrkA autotransporter, thus establishing a foundation for potential immunotherapies and tumour imaging strategies through further study of the BrkA-C7b anti-HER2 nanobody display system.