Navegando por Palavras-chave "combinatorial library"
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- ItemSomente MetadadadosEvaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities(Elsevier B.V., 2004-05-01) Campos, ITN; Silva, M. M.; Azzolini, S. S.; Souza, A. F.; Sampaio, CAM; Fritz, H.; Tanaka, A. S.; Universidade Federal de São Paulo (UNIFESP); Univ MunichA 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.
- ItemAcesso aberto (Open Access)Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors(Elsevier B.V., 1999-09-10) Tanaka, Aparecida Sadae [UNIFESP]; Silva, Melissa M. [UNIFESP]; Torquato, Ricardo Jose Soares [UNIFESP]; Noguti, Maria Aparecida Eiko [UNIFESP]; Sampaio, Claudio Augusto Machado [UNIFESP]; Fritz, H.; Auerswald, E. A.; Universidade Federal de São Paulo (UNIFESP); Univ MunichThe 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.
- ItemSomente MetadadadosHuman recombinant endopeptidase PHEX has a strict S-1 ' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein(Portland Press, 2003-07-01) Campos, Marcelo [UNIFESP]; Couture, Constance; Hirata, Izaura Y. [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Loisel, Thomas P.; Crine, Philippe; Juliano, Luiz [UNIFESP]; Boileau, Guy; Carmona, Adriana Karaoglanovic [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Montreal; BioMep IncThe PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. in order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S-1' subsite, with a strong preference for aspartate. Subsites S-2', S-1 and S-2 exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P-2', or valine, isoleucine or histidine in P, precluded hydrolysis of the substrate by the enzyme. the peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P-2 to P-2' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/K-m of 167 mM(-1) . s(-1). in addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N-(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity.