Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

Validation of a Phage Display Method for Protease Inhibitor Selection Using SFTI and HiTI Synthetic Hybrid Peptides

“…In an attempt to use HiTI mutant library as a tool to study trypsin-like enzymes specificities, the HiTI gene fragment was cloned into the phagemid pCANTAB 5E, so that it could be further used in a combinatorial mutant library, by displaying it on a phage surface. A functional display of HiTI on phage tips was expected, based on previous results of other Kunitz type inhibitors [26,42,43]. Our strategy for constructing the library was to conserve the Arg at P1 position based on trypsin specificity for basic amino acids in this position, and to restrict the number of possible mutations, the Leu at P4' position was also maintained, only the P1', P2', P3' and P5' were randomized to allow the construction of a representative library.…”

“…The display and selection of antibody chains on phage surfaces are the most important application of this technology [20,21]. In addition, phage display libraries have been used successfully to define enzyme substrates [22], to increase the anticoagulant activity of proteins [23,24], and to select specific protease inhibitors [25][26][27][28][29][30]. Previously, we evaluated the expression, display and selection of LDTI (Leech Derived Tryptase Inhibitor) variants specific for thrombin [29], human neutrophil elastase and human plasmin [25].…”

Engineered proteins as specific binding reagents

“…in [6]). More recent examples include the leech-derived trypsin inhibitor (LDTI) [63,64], the mustard trypsin inhibitor II (MTI II) [65,66] and ecotin [67 ].…”

“…Inhibition was restricted to thrombin and trypsin, while factor Xa, plasma kallikrein and neutrophil elastase were not inhibited. This study was further extended [64], and highly specific inhibitors to plasmin and neutrophil elastase were selected. To improve plant defense against aphids (soft-bodied insects), MTI II was subjected to phage display selection against trypsin and chymotrypsin [65,66].…”

Effect of invertebrate serine proteinase inhibitors on carrageenan-induced pleural exudation and bradykinin release

“…The inhibitors used to induce pleurisy were: LDTI-2PL, recombinant leech-derived tryptase inhibitor-2PL [21]; recombinant mutant aprotinin [22]; EIsynt, synthetic human neutrophil elastase inhibitor (Calbiochem-Novabiochem, CA, USA); BmTIs, Boophilus microplus trypsin inhibitors [23]. The proteinase inhibitors used in the radioimmunoassay were: E-64, trans-epoxysuccinyl-L-leucyl-amido-(4-guanidino)butane (Calbiochem-Novabiochem); EDTA, ethylenediaminetetracetic acid (Gibco BRL, Life Technologies, NY, USA); captopril (Sigma, MO, USA); SBTI, soybean trypsin inhibitor (Amersham Biosciences, Uppsala); aprotinin (kindly donated by Ennes A. Auerswald [22]); dexamethasone (DecadronR, Prodome, NJ, USA); and E-carrageenan (Sigma).…”