Mutation of the peptide-regulated transcription factor ComR for amidated peptide specificity and heterologous function in Lactiplantibacillus plantarum WCFS1.

There is a growing interest in the use of probiotic bacteria as biosensors for the detection of disease. However, there is a lack of bacterial receptors developed for specific disease biomarkers. Here, we have investigated the use of the peptide-regulated transcription factor ComR from Streptococcus spp. for specific peptide biomarker detection. ComR exhibits a number of attractive features that are potentially exploitable to create a biomolecular switch for engineered biosensor circuitry within the probiotic organism Lactiplantibacillus plantarum WCFS1. Through iterative design-build-test cycles, we developed a genomically integrated, ComR-based biosensor circuit that allowed WCFS1 to detect low nanomolar concentrations of ComR's cognate peptide XIP. By screening a library of ComR proteins with mutant residues substituted at the K100 position, we identified mutations that increased the specificity of ComR toward an amidated version of its cognate peptide, demonstrating the potential for ComR to detect this important class of biomarker.IMPORTANCEUsing bacteria to detect disease is an exciting possibility under active study. Detecting extracellular peptides with specific amino acid sequences would be particularly useful as these are important markers of health and disease (biomarkers). In this work, we show that a probiotic bacteria (Lactiplantibacillus plantarum) can be genetically engineered to detect specific extracellular peptides using the protein ComR from Streptococcus bacteria. In its natural form, ComR allowed the probiotic bacteria to detect a specific peptide, XIP. We then modified XIP to be more like the peptide biomarkers found in humans and engineered ComR so that it activated with this modified XIP and not the original XIP. This newly engineered ComR also worked in the probiotic bacteria, as expected. This suggests that with additional engineering, ComR might be able to activate with human peptide biomarkers and be used by genetically engineered probiotic bacteria to better detect disease.

Turning antibodies off and on again using a covalently tethered blocking peptide.

In their natural form, antibodies are always in an "on-state" and are capable of binding to their targets. This leads to undesirable interactions in a wide range of therapeutic, analytical, and synthetic applications. Modulating binding kinetics of antibodies to turn them from an "off-state" to an "on-state" with temporal and spatial control can address this. Here we demonstrate a method to modulate binding activity of antibodies in a predictable and reproducible way. We designed a blocking construct that uses both covalent and non-covalent interactions with the antibody. The construct consisted of a Protein L protein attached to a flexible linker ending in a blocking-peptide designed to interact with the antibody binding site. A mutant Protein L was developed to enable photo-triggered covalent crosslinking to the antibody at a specific location. The covalent bond anchored the linker and blocking peptide to the antibody light chain keeping the blocking peptide close to the antibody binding site. This effectively put the antibody into an "off-state". We demonstrate that protease-cleavable and photocleavable moieties in the tether enable controlled antibody activation to the "on-state" for anti-FLAG and cetuximab antibodies. Protein L can bind a range of antibodies used therapeutically and in research for wide applicability.