Abstract
Celiac disease is an autoimmune disorder characterized by damage to the small intestinal epithelium resulting from an immunogenic response to gluten. Patients with the disease may experience symptoms like diarrhea, fatigue, and nutrient malabsorption, highlighting that a strict gluten-free diet remains the only effective treatment. Subtilisin Carlsberg (SubC), a protease derived from Bacillus licheniformis, is capable of gluten degradation via its mature domain. A type V autotransporter system known as Bordetella resistance to killing A (BrkA) allows for surface expression of heterologous proteins. However, previous attempts to express the homologous subtilisin E in Escherichia coli using the BrkA type V autotransporter system were unsuccessful, likely due to the misfolding of the mature domain. In subtilisin-family proteases, proper folding is mediated by an N-terminal pro domain acting as a chaperone. Here, we propose the BrkA-mediated surface expression of SubC in E. coli, a common gut commensal, using a trans-complementation system. The pro (pCARP) and mature (pCARM) domains will be expressed on separate plasmids in the OmpT-deficient E. coli UT5600 strain. We hypothesize that the trans-complementation of these domains will enable proper folding and proteolytic activity of the mature domain. In our project, we successfully assembled the pCARM plasmid and confirmed the surface expression using a trypsin accessibility assay. However, assembly of the pCARP plasmid remained incomplete, preventing functional validation of the dual-plasmid system. This study explores pro-domain-assisted mature domain folding in-trans as a strategy for functional enzyme display, laying the groundwork for future therapeutic approaches for celiac disease. Future work should focus on completing the pCARP assembly and evaluating proteolytic activity under pH conditions representative of the small intestine to evaluate in vivo feasibility. This system could enable localized gluten degradation in the gut, improving health outcomes and quality of life for individuals with celiac disease.