Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors
Research article published in FEBS letters (1999)
Abstract
The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced 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 (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin 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 thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage.
Por qué esto importa para la hirudoterapia
Este estudio utilizó presentación de fagos funcional para exponer el inhibidor de triptasa derivado de la sanguijuela (LDTI) en superficies del fago M13 y creó una biblioteca combinatoria de aproximadamente 52 000 mutantes que varían en las posiciones del sitio reactivo P1–P4'. Tras la selección contra trombina, se produjeron tres variantes en levaduras: LDTI-2T y LDTI-5T inhibieron la trombina (Ki de 302 nM y 28 nM, respectivamente) y prolongaron el tiempo de coagulación sanguínea, mientras que LDTI-10T se unió pero no inhibió la trombina. Los resultados revelaron una fuerte preferencia por arginina en P1 y valina en P1' para la inhibición de la trombina, identificándose a LDTI-5T como un candidato modelo para generar inhibidores de trombina dirigidos al sitio activo. Esto es relevante para el dominio de ASH ya que diseña directamente un andamiaje inhibidor derivado de la sanguijuela para una nueva actividad inhibitoria de trombina. La advertencia es que se trata de un estudio de ingeniería molecular in vitro sin datos animales ni clínicos reportados en el resumen.
Citación
Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors
Tanaka AS et al. · FEBS letters, 1999
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026