Molecular Weight-based Concentration and Ni-NTA Enrichment Enhance Detection of OmpT-cleaved BrkA Passenger Domain in the Extracellular Milieu of Escherichia coli UT2300

Autotransporter secretion systems are used by Gram-negative bacteria to transport proteins across the cell envelope. Bordetella resistance to killing protein A (BrkA) is an autotransporter protein in Bordetella pertussis whose passenger domain remains non-covalently associated with the cell surface following translocation across the outer membrane. The surface-retention property of its passenger domain has enabled BrkA to be exploited for various biotechnological applications. However, this property limits its potential for use in applications that require soluble protein secretion. To overcome this limitation, previous work engineered an exogenous outer membrane protease T (OmpT) cleavage site into the BrkA passenger domain to promote its release. Although successful cleavage was observed, the passenger domain was not detected in the extracellular milieu of the Escherichia coli expression host via Western blot analysis. This may be due to the inherently low secretion efficiency of E. coli for heterologous proteins, resulting in extracellular levels below the detection limit of conventional detection assays. In this study, we employed molecular weight cut-off concentration and nickel-nitrilotriacetic acid affinity enrichment to enable the detection of low-abundance OmpT-cleaved BrkA passenger domain in the extracellular milieu of OmpT+ E. coli UT2300 cells. We observed a strong Western blot signal corresponding to the OmpT-cleaved BrkA passenger domain in the extracellular milieu following the application of molecular weight cut-off concentration alone, and after nickel-nitrilotriacetic acid affinity purification and molecular weight cut-off concentration were applied together. These results demonstrate that combining molecular weight cut-off with nickel-nitrilotriacetic acid affinity enrichment enables the detection of low-abundance OmpT-cleaved BrkA passenger domain and highlights a useful strategy for the detection and purification of secreted autotransporter-derived proteins.