Preliminary Steps Towards the Construction of a Customizable BrkA Protein Secretion System

Abstract

Type Va autotransporters, such as BrkA in Bordetella pertussis, offer a unique platform for the surface display of diverse proteins. The BrkA passenger domain can be modified to display proteins in Escherichia coli's outer membrane. However, existing methods retain a substantial portion of the passenger domain. Here, we propose a potential design for an adaptable plasmid for surface protein expression known as PassOut. PassOut is a modifiable bacterial surface display and detection system, achieved by removing 1674 nucleotides of the BrkA passenger domain (Q43-P600) and replacing it with a multiple cloning site (MCS) encoding a C-terminal 6X-histidine tag (His-tag) and unique EcoRI and NdeI restriction enzyme cleavage sites. The PassOut system aims for a more compact BrkA passenger size and includes the auto-chaperone region required for protein folding and secretion. Design details including the use of synthetic DNA sequences, PCR, and Gibson assembly are outlined. Our results demonstrate amplification of the DNA insert encoding the MCS and restriction enzyme cleavage sites, and linearization of the plasmid pKAX5A backbone. However, we were unable to obtain recombinant plasmids encoding the desired PassOut insert sequence after two cloning attempts using Gibson cloning technology. It is possible that the introduction of non-native amino acids encoded in the PassOut DNA sequence resulted in a toxic form of BrkA that prevented the growth of colonies. It is suggested that future studies target another site in pKAX5A for the introduction of an MCS.