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.
Резюме
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.
Почему это важно для гирудотерапии
This study used functional phage display to present the leech-derived tryptase inhibitor (LDTI) on M13 phage surfaces and created a combinatorial library of approximately 52,000 mutants varying at the P1–P4' reactive-site positions. After selection against thrombin, three variants were produced in yeast: LDTI-2T and LDTI-5T inhibited thrombin (Ki 302 nM and 28 nM, respectively) and prolonged blood clotting time, while LDTI-10T bound but did not inhibit thrombin. The results revealed a strong preference for arginine at P1 and valine at P1' for thrombin inhibition, with LDTI-5T identified as a model candidate for generating active-site-directed thrombin inhibitors. This is relevant to ASH's domain as it directly engineers a leech-derived inhibitor scaffold for new thrombin-inhibitory activity. The caveat is that this is an in vitro molecular engineering study with no animal or clinical data reported in the abstract.
Цитирование
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
Связанный клинический контекст
Узнайте, как это исследование связано с клинической практикой
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