Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae.
Mol Biochem Parasitol
; 88(1-2): 163-74, 1997 Sep.
Article
en En
| MEDLINE
| ID: mdl-9274877
ABSTRACT
Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.
Buscar en Google
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Endopeptidasas
/
Catepsinas
/
ADN Complementario
/
ADN de Helmintos
/
Fasciola hepatica
Límite:
Animals
Idioma:
En
Revista:
Mol Biochem Parasitol
Año:
1997
Tipo del documento:
Article
País de afiliación:
Irlanda