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

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

Author Tanaka, Aparecida Sadae Autor UNIFESP Google Scholar
Silva, Melissa M. Autor UNIFESP Google Scholar
Torquato, Ricardo Jose Soares Autor UNIFESP Google Scholar
Noguti, Maria Aparecida Eiko Autor UNIFESP Google Scholar
Sampaio, Claudio Augusto Machado Autor UNIFESP Google Scholar
Fritz, H. Google Scholar
Auerswald, E. A. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Univ Munich
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 (K-i 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. (C) 1999 Federation of European Biochemical Societies.
Keywords phage display system
thrombin inhibitor
combinatorial library
Kazal-type serine proteinase inhibitor
filamentous phage
Language English
Date 1999-09-10
Published in Febs Letters. Amsterdam: Elsevier B.V., v. 458, n. 1, p. 11-16, 1999.
ISSN 0014-5793 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 11-16
Origin http://dx.doi.org/10.1016/S0014-5793(99)01106-0
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000082617200003
URI http://repositorio.unifesp.br/handle/11600/26142

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