(513e) Profiling Protease Substrate Specificity with Yess-NGS

Proteases are important effectors of numerous physiological and pathological processes. In depth-determination of protease specificity is vital to understand protease function and to design activity-based probes and inhibitors. Moreover, engineered protease therapeutics with altered specificities require that their substrate specificity also be profiled in order to gain insight into their in vivomechanism. While various chemical and biological and proteomics-based approaches, including microarray, phage display, and bacterial display have been developed to characterize protease substrate specificity, none combine a protease engineering platform with a comprehensive substrate specificity profiling technology.

Previously, we developed the yeast endoplasmic reticulum (ER) sequestration screening (YESS) system for engineering and profiling substrate specificity. In this report, we combine YESS with Next-Gen sequencing (YESS-NGS) to enable a comprehensive survey of protease specificity. In YESS-NGS, a genetically-encoded combinatorial peptide library transits through the yeast ER where it interacts with a recombinant protease residing in the ER before being transported through the secretory pathway to the cell surface. Since the combinatorial library is flanked by epitope tags, substrate peptide cleavage can be detected with fluorophore-conjugated antibodies and cells exhibiting the desired “cleaved” phenotype can be sorted by multicolor FACS. Subsequently, DNA recovered from such cells are barcoded and submitted for NGS to profile the cleaved substrates. Previously, we used YESS-NGS to profile proteolysis of the yeast secretory pathway, revealing a major cleavage pattern effected by the endogenous protease kex2. This led us to generate the protease profiling strain EBY100^kex2-, an improved strain for protease profiling in which known kex2 substrates are no longer removed from the substrate pool. Using EBY100^kex2, we profiled several proteases including kallikreins 1, 2, 3, and 6, the insulin-degrading enzyme (IDE), human elastase and engineered variants.