Abstract
The Type V autotransporter system, exemplified by Bordetella resistance to killing (BrkA) from Bordetella pertussis, has been used as a platform for the surface display of heterologous proteins. While many autotransporters contain an internal autochaperone domain to facilitate folding of the passenger domain, its necessity for correct folding and function of heterologous passenger domains, such as β-lactamase is not yet studied. In this study, we investigated the role of BrkA autochaperone domain in the stability of ꞵ-lactamase passenger in Escherichia coli strain UT5600 using two engineered constructs: pBLacIn containing the autochaperone domain, and pBLacSwitch which lacks the autochaperone domain. Initial analysis of native BrkA revealed a cleaved passenger domain showing resistance to trypsin concentrations up to 15 μg/mL. Growth assays in the presence of Penicillin G were conducted in order to assess the functionality of ꞵ-lactamase without an autochaperone domain. There was no significant difference in growth between pBLacIn and pBLacSwitch, while the BrkA control consistently reached a lower peak OD600. Western blot analysis of pBLacIn and pBLacSwitch treated with 15 μg/mL showed an absence of detectable protein expression with and without trypsin, possibly due to their dimerization. These findings suggest that the BrkA autochaperone domain may not be necessary for promoting correct folding and function of ꞵ-lactamase passenger protein, however plasmid dimerization may be impacting these results.