Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors

The recombinant phage antibody system pCANTAB 5E has been used to display f unctionally active leech-derived tryptase inhibitor (LDTI) on the tip of th e filamentous M13 phage, A limited combinatorial library of 5.2 x 10(4) mut ants was created with a synthetic LDTI gene, using a degenerated oligonucle otide and the pCANTAB 5E phagemid. The mutations were restricted to the P1- P4' positions of the reactive site. Fusion phages and appropriate host stra ins containing the phagemids were selected after binding to thrombin and DN A sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8 R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were prod uced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (K-i 6.4 nM and 2.1 nM) but not factor Xa, plasm a kallikrein or neutrophil elastase, The variant LDTI-10T binds to thrombin but does not inhibit it, The relevant reactive site sequences of the throm bin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V), The data indicate further that LDTI- 5T might be a model candidate for generation of active-site directed thromb in inhibitors and that LDTI in general may be useful to generate specific i nhibitors suitable for a better understanding of enzyme-inhibitor interacti ons. (C) 1999 Federation of European Biochemical Societies.