Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors
Autor: | Hans Fritz, Ennes A. Auerswald, Aparecida S. Tanaka, Melissa Andreia de Moraes Silva, Claudio A. M. Sampaio, Ricardo J.S. Torquato, Maria Aparecida Eiko Noguti |
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Rok vydání: | 1999 |
Předmět: |
Phage display
Phagemid Mutant Molecular Sequence Data Biophysics Saccharomyces cerevisiae Biology Biochemistry law.invention Thrombin Chymases Structural Biology law Peptide Library Genetics medicine Humans Bacteriophages Amino Acid Sequence Cloning Molecular Molecular Biology Base Sequence Dose-Response Relationship Drug Models Genetic Oligonucleotide Serine Endopeptidases Proteins Cell Biology Trypsin Molecular biology Kinetics Mutagenesis Insertional Recombinant DNA Tryptases medicine.drug Discovery and development of direct thrombin inhibitors |
Zdroj: | FEBS letters. 458(1) |
ISSN: | 0014-5793 |
Popis: | 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 5.2×104 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 (Ki 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. |
Databáze: | OpenAIRE |
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