Investigating the Influence of the BrkA Auto chaperone Region on Recombinant Protein Surface Expression and Activity in Escherichia coli

Bacterial surface display systems represent a promising new avenue for biotechnology, enabling protein or peptide presentation. Type V autotransporters are Gram-negative bacterial secretion systems that present their passenger domain across the outer cell membrane. These intrinsic bacterial display systems bypass challenges associated with secreted proteins, such as the need for purification. The Bordetella resistance to killing A protein (BrkA) type V secretion system, from Bordetella pertussis, has previously been used for surface display of heterologous proteins. One region of the BrkA passenger domain, the auto chaperone, has previously been implicated in the proper folding and secretion of the endogenous passenger, however its role in secretion of exogenous, or non-native, passengers has yet to be investigated. Here, we investigated the requirement of the BrkA auto chaperone for surface display of the antibiotic resistance enzyme ß-lactamase. We generated recombinant protein constructs in which the endogenous BrkA passenger domain was replaced with ß-lactamase, either retaining or deleting the auto chaperone domain. Following transformation into Escherichia coli, we determined that the auto chaperone domain is not required for surface expression of heterologous ß-lactamase-BrkA recombinant proteins, and that expression of these constructs does not affect E. coli growth kinetics. This study demonstrates the first instance of heterologous BrkA auto-display without the auto chaperone. Further investigation into the functionality of the secreted ß-lactamase is of interest for delineating the specific requirements of BrkA-mediated surface display in the context of downstream applications.