Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties.

Détails

ID Serval
serval:BIB_0C1BEB907AB1
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties.
Périodique
Journal of Biological Chemistry
Auteur(s)
Lin X., Tang J., Koelsch G., Monod M., Foundling S.
ISSN
0021-9258
Statut éditorial
Publié
Date de publication
09/1993
Peer-reviewed
Oui
Volume
268
Numéro
27
Pages
20143-20147
Langue
anglais
Résumé
A cDNA fragment which encodes the zymogen of canditropsin, the extracellular aspartic protease from the yeast Candida tropicalis (Togni,G., Sanglard, D., Falchetto, R., and Monod, M. (1991) FEBS Lett. 286, 181-185) was cloned into a T7 expression vector for the synthesis of the recombinant zymogen in Escherichia coli. Recombinant canditropsinogen (Ctg), which was expressed as inclusion bodies in the cytosol of E. coli, was refolded by dialysis from an 8 M urea solution and purified to homogeneity using chromatographies on Sephacryl S-300 and on MonoQ columns. The purified Ctg was converted into canditropsin by either acid activation or trypsin conversion. The specificity of the resulting recombinant canditropsin toward polypeptide substrates is significantly different from other aspartic proteases. Canditropsin hydrolyzes oxidized insulin B chain between Ala-Leu and many other minor cleavage sites. Canditropsin also hydrolyzes keratin and collagen, which are components of connective tissues known to be hydrolyzed by canditropsin during Candida infections. Canditropsin was strongly inhibited by the universal aspartic protease inhibitor pepstatin (Ki = 1.75 x 10(-8) M) and inactivated by two aspartic protease inactivators, DAN and EPNP. Canditropsin is weakly inhibited by leupeptin and antipain, with an apparent Ki of 1.74 x 10(-4)M and 1.5 x 10(-5) M, respectively.
Mots-clé
Amino Acid Sequence, Aspartic Endopeptidases/biosynthesis, Aspartic Endopeptidases/isolation & purification, Base Sequence, Candida/enzymology, Candida/genetics, Chromatography, High Pressure Liquid, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Escherichia coli/genetics, Gene Expression, Genetic Vectors, Hydrogen-Ion Concentration, Insulin/metabolism, Kinetics, Molecular Sequence Data, Molecular Weight, Oligodeoxyribonucleotides, Peptide Fragments/isolation & purification, Polymerase Chain Reaction, Protease Inhibitors/pharmacology, Recombinant Proteins/biosynthesis, Recombinant Proteins/isolation & purification, Substrate Specificity
Pubmed
Web of science
Création de la notice
25/01/2008 16:47
Dernière modification de la notice
20/08/2019 12:33
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