Please use this identifier to cite or link to this item:
|Title:||Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors|
|Authors:||Tanaka, Aparecida Sadae [UNIFESP]|
Silva, Melissa M. [UNIFESP]
Torquato, Ricardo Jose Soares [UNIFESP]
Noguti, Maria Aparecida Eiko [UNIFESP]
Sampaio, Claudio Augusto Machado [UNIFESP]
Auerswald, E. A.
Universidade Federal de São Paulo (UNIFESP)
|Keywords:||phage display system|
Kazal-type serine proteinase inhibitor
|Citation:||Febs Letters. Amsterdam: Elsevier B.V., v. 458, n. 1, p. 11-16, 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 (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.|
|Appears in Collections:||Artigo|
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.