Purification and characterization of active recombinant rat kallikrein rK9

Purification and characterization of active recombinant rat kallikrein rK9

Autor Zani, M. L. Google Scholar
Brillard-Bourdet, M. Google Scholar
Lazure, C. Google Scholar
Juliano, L. Google Scholar
Courty, Y. Google Scholar
Gauthier, F. Google Scholar
Moreau, T. Google Scholar
Instituição Univ Tours
Clin Res Inst Montreal
Universidade Federal de São Paulo (UNIFESP)
Resumo The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast a-factor cDNA. the fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase, Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. the identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. the dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K-m) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K-m of 420 000 M-1 s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9. (C) 2001 Elsevier Science B.V. All rights reserved.
Assunto recombinant protease
tissue kallikrein
enzyme kinetics
Pichia pastoris expression
Idioma Inglês
Data 2001-06-11
Publicado em Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology. Amsterdam: Elsevier B.V., v. 1547, n. 2, p. 387-396, 2001.
ISSN 0167-4838 (Sherpa/Romeo, fator de impacto)
Editor Elsevier B.V.
Extensão 387-396
Fonte http://dx.doi.org/10.1016/S0167-4838(01)00208-4
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000169535700022
URI http://repositorio.unifesp.br/handle/11600/26581

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