Abstract
β-Hexachlorocyclohexane (β-HCH) is a persistent environmental pollutant that resists degradation and accumulates in the ecosystem, posing risks to human health. Haloalkane dehalogenases such as LinB from Sphingobium species offer potential for bioremediation, but their intracellular localization limits access to extracellular substrates. To address this, we investigated the BrkA autotransporter system of Bordetella pertussis for surface display and potential protease-mediated secretion of LinB in Escherichia coli. The previously developed pITAS construct encodes a BrkA-LinB fusion protein containing an engineered OmpT cleavage site and an N-terminal 6xHis-tag, and was expressed in both E. coli UT5600 (OmpT-) and UT2300 (OmpT+) strains. Subcellular fractionation determined that BrkA-LinB localized predominantly to the outer membrane in both E. coli strains, with bands at ~170-180 kDa corresponding to the full-length protein. Trypsin accessibility assay showed loss of those bands upon treatment, further confirming surface display. In the OmpT+ strain, no His-tagged BrkA-LinB cleavage products were detected in the concentrated supernatant. In contrast, the positive control pOCS produced a ~65 kDa band in the supernatant, consistent with OmpT-mediated cleavage. These findings indicate that while BrkA supports surface display of LinB, OmpT-mediated cleavage and extracellular release were not observed in the pITAS construct. Given that surface display was confirmed in pITAS but cleavage products were only detected in pOCS, this suggests that insertion of LinB into the BrkA passenger domain may be affecting the accessibility of the engineered cleavage site.