Proteases represent an important class of enzymes in all eukaryotic cells where they play critical roles in activating and destroying proteins during cell growth, development, cell signaling, apoptosis, and autophagy. Misregulation, however, can lead to disease; for example, cancer cells often secrete metalloproteases that degrade the extracellular matrix, permitting cancers cells to metastasize and take root at various locations in the body. Proteases work by recogniz- ing linear regions, typically 6 to 8 residues, in their substrates and hydrolyzing a peptide bond, thereby fragmenting their substrate into N-terminal and C-terminal fragments. Understanding the range of peptide sequences cleaved by a protease, termed the "proteolysis signature" or "substrate profile", has many scientific implications and practical benefits. One powerful method for determining the proteolysis signature or substrate profile of a recombinant protease is through the use of combinatorial peptide libraries. In this effort, there are two specific aims. In Aim #1, we will develop an improved phage vector for protease site identification by allowing for more efficient cleavage of recognized sequences, more even representation of amino acids, and identification of longer recognition sequences (up to 10 AA).The vectors will carry the kanamycin resistance gene and a second copy of gene III that is tagged at the N-terminus with the AviTag and has the same cloning site cassette at three different locations. After the vectors have been demonstrated to be suitable as substrates for two model proteases, oligonucleotides will be ordered with a 10-mer region randomized with 19 trinucleotide phosphoramidites at equal frequency encoding 19 amino acids (excluding cysteine) for cloning into each site and large (> 1010) libraries created. The OmpT gene in the bacterial strain, XL1-Blue, will be knocked out by homologous recombination to eliminate the loss of phage-displayed peptides in the libraries that carry dibasic residues. In Aim #2, Tango will evaluate the utility of the new libraries with two commercially available proteases, the human metalloprotease, ADAM15, and the Escherichia coli protease, DegP. Virions will be captured on neutravidin-coated magnetic beads, released with each protease, and the output will be sequenced by Next Generation Sequencing (NGS). Nimble Therapeutics will then synthesize two million peptides on arrays, corresponding to 10 or more copies of each peptide sequence discovered by NGS, along with systematic amino acid replacements of any motifs identified among the selected peptides. The peptides will carry a peptide tag at their C-termini, which binds to fluorescent streptavidin, and a PEG linker at their N-termini to distance them it away from the array surface. Arrays will be incubated with the pro- tease, incubated with fluorescent streptavidin, and scans will reveal which spots lose fluores- cence due to proteolysis. To confirm that such peptides are t...