Design and characterization of a new pVII combinatorial phage display peptide library for protease substrate mining and using Factor VII activating protease (FSAP) as a model

We describe a novel, easy and efficient combinatorial phage display peptide substrate mining method to map the substrate specificity of proteases. The peptides are displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, unmodified. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any posttranslational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss of function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified, and indeed one of these peptides were cleaved as a free peptide suggesting a broader range of WT-FSAP substrates than previously anticipated.,© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.