Peptide gene sequences of substrate phage for different proteases

We present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical 6 to 8 residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases 2, 3, 6, 7 and 8, and the ectodomains of the membrane sheddases, ADAMs 10 and 17. The first library (Lib 10AA) was used to determine substrate cleavage motifs. Lib 10AA contains a highly diverse randomized 10-mer substrate peptide sequences (10^9 unique members) that was displayed mono-valently on filamentous phage and bound to magnetic beads via an N-terminal biotin. The protease was allowed to cleave the SPD beads, and the released phage subjected to up to three total rounds of positive selection followed by next generation sequencing (NGS). This allowed us to identify from 10^4 to 10^5 unique cleavage sites over a broad dynamic range of NGS counts (ranging from 3-5000), and produced consensus and optimal cleavage motifs based positional sequencing scoring matrices and state-of the-art machine learning algorithm that closely matched synthetic peptide data. A second SPD-NGS library (Lib hP) was constructed that allowed us to identify candidate human proteome sequences. Lib hP displayed virtually the entire human proteome tiled in contiguous 49AA sequences with 25AA overlaps (nearly 1 million members). After three rounds of positive selection we identified up to 10^4 natural linear cut sites depending on the protease and captured most of the examples previously identified by proteomics (ranging from 30 to 1000) and predicted 10 to 100-fold more. Overall design: To perform a SPD-NGS screening, 1 mL of biotinylated substrate phage library was incubated with 100 µL magnetic StreptAvidin (SA) beads for 30 min and after that, the SA beads were then stringently washed with PBS buffer supplemented with 0.05% Tween20 and 0.2% BSA to get rid of any free phage. After the SA beads were treated with protease of interest, released phage in supernatant were collected and propagated as protease-sensitive pool, and those bound on the beads can also be propagated as protease-resistant pool after released by non-specific proteases like trypsin. After three rounds of selection, phage polymerase chain reaction (PCR) was conducted, followed by NGS to identify sensitive substrate sequences in each pool. We tested 7 proteases and generated 21 x 2 datasets (3 rounds of selection for each protease).