Construction of a Plasmid that Expresses an N-terminally 6xHis Tagged Protein Spanning Amino Acids Gly139 to Pro552 of the Passenger Domain of the Escherichia coli Autotransporter Protein Ag43

Abstract

Escherichia coli , a Gram-negative bacterium, exhibits resistance to multiple antibiotics, emphasising the need for novel targets. Autotransporter (AT) proteins, such as Antigen 43 (Ag43), have emerged as potential targets due to their association with virulence. Autotransporters are a family of secreted proteins in Gram-negative bacteria, and Ag43, with its modular structure, plays a prominent role. This study focuses on the secreted passenger domain of Ag43. Previous research suggested that the autochaperone, a conserved amino acid sequence located at the C-terminus of many autotransporter passengers, is required for proper passenger domain folding during secretion. Our goal was to investigate the role of the so called autochaperone domain in the Ag43 passenger sequence. Our study aimed to create recombinant plasmids that would enable expression, purification, and refolding of Ag43 passenger domain variants with and without the putative autochaperone region. Using PCR and standard cloning techniques, we were able to create a plasmid termed pXYZ encoding an N-terminally tagged Ag43 passenger lacking the autochaperone region (amino acid X to amino acid Y). We were unable to create a plasmid encoding Ag43 containing the autochaperone region.  Follow-up analyses using SDS PAGE showed that Ag43 (lacking the autochaperone region) is expressed in the cytosol of E. coli strain BL21. This project lays the building blocks for investigating the role of the autochaperone region in Ag43 folding and the effector function of Ag43 in order to characterise the motif as a potential novel antimicrobial target.