Abstract
Autotransporter-based surface display systems, such as the Bordetella pertussis autotransporter (BrkA), have been widely utilized as a platform for presenting heterologous proteins on the surface of Gram-negative bacteria, making it a useful scaffold for bacterial display applications. The Ty1 nanobody is a small alpaca-derived antibody fragment that binds the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain with high affinity and blocks angiotensin-converting enzyme 2 (ACE2) interaction, making it a relevant candidate for diagnostic and therapeutic development. Although nanobodies fused to the BrkA scaffold have been expressed intracellularly before, there is no existing evidence of its ability to be surface displayed while maintaining proper folding. In this study, we investigated whether BrkA could be used as a scaffold for the surface display of the Ty1 nanobody while maintaining evidence of proper folding. We constructed pTHNX, a pPALMC1-derived plasmid containing the Ty1 coding sequence inserted into the BrkA passenger domain and verified the construct by colony PCR and subsequent sequencing. The recombinant plasmid construct was transformed into Escherichia coli UT5600 and Western blot analysis confirmed intracellular expression of BrkA-Ty1 fusion construct. Protein surface display and folding were evaluated using trypsin accessibility and susceptibility assays. Results from these assays indicated that the fusion construct was surface displayed, although susceptibility to low concentrations of trypsin suggested improper folding of the Ty1 nanobody. These findings demonstrate the potential of BrkA as a bacterial display platform for nanobody engineering and highlight key challenges associated with maintaining proper folding and functionality of heterologous proteins, especially nanobodies, during autotransporter-mediated presentation at the cell surface.