Characterizing Surface Display of a BrkA Fusion Protein Using a Trypsin Accessibility Assay

A trypsin accessibility assay offers a direct approach for assessing surface display because externally exposed regions of a Bordetella pertussis autotransporter (BrkA) fusion protein are susceptible to proteolytic cleavage, whereas inaccessible or improperly displayed proteins remain protected. Here, we describe a detailed protocol for using a trypsin accessibility assay to characterize surface display of a BrkA fusion protein expressed in Escherichia coli UT5600. The protocol includes controlled trypsin treatment of intact cells, sample preparation for SDS-PAGE, and immunodetection of cleavage products by Western blot using an anti-6xHis antibody. Surface-displayed proteins are expected to remain largely intact, allowing clear discrimination between exposed and inaccessibly fusion proteins. This assay provides an accessible and reproducible strategy for evaluating BrkA-mediated surface presentation of recombinant proteins and may be adapted for characterization of other autotransporter-based display constructs, thus supporting broader application of autotransporter systems in protein engineering and surface display studies.