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

FEBS Letters

Torquato, Ricardo J.S.²  Silva, Melissa M.²  Sampaio, Claudio A.M.²  Auerswald, Ennes A.¹  Noguti, Maria A.E.³  Fritz, Hans¹  Tanaka, Aparecida S.² 

[1] Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt der Ludwig-Maximilians Universität, Munich, Germany;Departamento de Bioquı́mica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil;Departamento de Medicina, Disciplina de Hematologia, UNIFESP-EPM, São Paulo, SP, Brazil

关键词: Phage display system;    Thrombin inhibitor;    Combinatorial library;    Kazal-type serine proteinase inhibitor;    Filamentous phage;    LDTI;    leech-derived tryptase inhibitor;    pIII;    minor coat protein III;    ELISA;    enzyme-linked immunosorbent assay;    moi;    multiplicity of infection;    cfu;    colony forming unit;    PCR;    polymerase chain reaction;    Amp;    ampicillin;    Kan;    kanamycin;    TT;    thrombin time (clotting time of human blood triggered by thrombin);   

DOI  :  10.1016/S0014-5793(99)01106-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 (K _i 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.

【 授权许可】

Unknown