Bacterial display of self-blocking affinity proteins for efficient protease substrate profiling
(English)Manuscript (preprint) (Other academic)
Proteases are involved in fundamental biological processes and are important tools in both industry and biomedical research. One of the most important mechanisms of a protease is its ability to discriminate among potential substrates. Here, we present a new method for substrate profiling of proteases. The substrates are displayed within a fusion protein on the surface of the Gram-positive bacteria S. carnosus. The substrate is inserted in a linker, which connects two anti-idiotypic Affibody molecules (ZHER2 and anti-ZHER2). ZHER2 has affinity for the HER2 receptor and anti-ZHER2 binds to ZHER2, thereby blocking the binding surface from interacting with HER2. By site-specific proteolysis of the substrate within the linker, the blocking domain is released and will diffuse away, thus the HER2 binding capacity of ZHER2 is restored. The proteolysis is therefore reflected in HER2 binding, which can be analyzed by flow cytometry upon labeling with fluorescent HER2. By applying this methodology we could enrich for cells displaying a substrate peptide, efficiently hydrolyzed by tobacco etch virus protease (TEVp), from a library of cells displaying different substrates. In an attempt to determine the substrate preference of matrix metalloprotease-1 (MMP-1), cells displaying a previously reported motif (PXXXHy) were enriched. On-cell determination of apparent kcat/KM revealed that the enriched substrate peptides were hydrolyzed 6-8 times more efficiently than a previously reported substrate peptide.
Protease, substrate profiling, bacterial display, combinatorial protein engineering, staphylococcal display, directed evolution, cell surface display, flow cytometry, MMP-1, tobacco etch virus protease, TEVp, HER2
Biochemistry and Molecular Biology
Research subject Biotechnology
IdentifiersURN: urn:nbn:se:kth:diva-166537OAI: oai:DiVA.org:kth-166537DiVA: diva2:811677
QS 201505212015-05-122015-05-112015-05-21Bibliographically approved