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

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dc.contributor.author Tanaka, Aparecida Sadae [UNIFESP]
dc.contributor.author Silva, Melissa M. [UNIFESP]
dc.contributor.author Torquato, Ricardo Jose Soares [UNIFESP]
dc.contributor.author Noguti, Maria Aparecida Eiko [UNIFESP]
dc.contributor.author Sampaio, Claudio Augusto Machado [UNIFESP]
dc.contributor.author Fritz, H.
dc.contributor.author Auerswald, E. A.
dc.date.accessioned 2016-01-24T12:30:53Z
dc.date.available 2016-01-24T12:30:53Z
dc.date.issued 1999-09-10
dc.identifier http://dx.doi.org/10.1016/S0014-5793(99)01106-0
dc.identifier.citation Febs Letters. Amsterdam: Elsevier B.V., v. 458, n. 1, p. 11-16, 1999.
dc.identifier.issn 0014-5793
dc.identifier.uri http://repositorio.unifesp.br/handle/11600/26142
dc.description.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. en
dc.format.extent 11-16
dc.language.iso eng
dc.publisher Elsevier B.V.
dc.relation.ispartof Febs Letters
dc.rights Acesso aberto
dc.subject phage display system en
dc.subject thrombin inhibitor en
dc.subject combinatorial library en
dc.subject Kazal-type serine proteinase inhibitor en
dc.subject filamentous phage en
dc.title Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors en
dc.type Artigo
dc.rights.license http://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dc.contributor.institution Universidade Federal de São Paulo (UNIFESP)
dc.contributor.institution Univ Munich
dc.description.affiliation UNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, Brazil
dc.description.affiliation Univ Munich, Klinikum Innenstadt, Chirurg Klin & Poliklin, Klin Chem & Klin Biochem Abt, D-8000 Munich, Germany
dc.description.affiliation UNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifesp UNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifesp UNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, Brazil
dc.identifier.file WOS000082617200003.pdf
dc.identifier.doi 10.1016/S0014-5793(99)01106-0
dc.description.source Web of Science
dc.identifier.wos WOS:000082617200003



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