The Dibasics of M13 phage display: examining the substrate specificity of Escherichia coli outer membrane proteases

Bacteriophage M13 substrate libraries are a useful tool to discover novel protease substrates. In this study, we have mapped 7-mer protease substrates cleaved by native Escherichia coli proteases during phage outgrowth. Cleavage was monitored by the loss of a biotinylated AviTag at the N-terminus of the recombinant M13 minor coat protein, protein III. Using this method, we isolated 120 phage-displayed substrates that contain dibasic amino acid motifs (Lys-Lys, Arg-Arg, Lys-Arg, and Arg-Lys), of which the Lys-Lys motif has not been previously described using substrate phage. Since dibasic motifs are known to be substrates of the outer membrane protein proteases OmpP and OmpT, the phage library was transferred into a mutant strain deficient in both protease activities, and the selection process was repeated. Sequencing of recovered clones revealed no enrichment of the four dibasic motifs, demonstrating that all four motifs are substrates of Omptin proteases. These findings indicate that phage propagation in Omptin-expressing bacteria leads to the loss of dibasic amino acid motifs from phage-displayed combinatorial peptide, protein, and antibody libraries. This suggests that the presence of dibasic residues in proteins and peptides displayed on bacteriophage M13 should necessitate the use of OmpT- and OmpP-deficient bacterial hosts.