Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities

Author Campos, ITN Google Scholar
Silva, M. M. Google Scholar
Azzolini, S. S. Google Scholar
Souza, A. F. Google Scholar
Sampaio, CAM Google Scholar
Fritz, H. Google Scholar
Tanaka, A. S. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Univ Munich
Abstract A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed oil the pCANTAB 5E phagemid, and LDTI fusion pliages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. the LDTI variants: 29E (K8A, 19A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2P1 (K11W! and P12N), SP1 (19V K11W, and P12E), and ION (19T K11L and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2P1, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor (K-i 0.5 W), affecting no other tested enzymes. LDTI-2P1 was the strongest plasmin inhibitor (K-i = 1.7 nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), front a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies. (C) 2004 Elsevier Inc. All rights reserved.
Keywords phage display system
neutrophil elastase inhibitor
plasmin inhibitor
combinatorial library
Kazal-type serine proteinase inhibitor
filamentous phage
Language English
Date 2004-05-01
Published in Archives of Biochemistry and Biophysics. New York: Elsevier B.V., v. 425, n. 1, p. 87-94, 2004.
ISSN 0003-9861 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 87-94
Origin http://dx.doi.org/10.1016/j.abb.2004.03.004
Access rights Closed access
Type Article
Web of Science ID WOS:000221003100010
URI http://repositorio.unifesp.br/handle/11600/27725

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