Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae
| Autor: | A J, Dowd, J, Tort, L, Roche, T, Ryan, J P, Dalton |
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| Rok vydání: | 1997 |
| Předmět: |
Enzyme Precursors
DNA Complementary Base Sequence Cathepsin L Molecular Sequence Data Fibrinogen Gene Expression Saccharomyces cerevisiae DNA Helminth Fasciola hepatica Hydrogen-Ion Concentration Cathepsins Polymerase Chain Reaction Recombinant Proteins Substrate Specificity Cysteine Endopeptidases Endopeptidases Animals Genes Helminth DNA Primers |
| Zdroj: | Molecular and biochemical parasitology. 88(1-2) |
| ISSN: | 0166-6851 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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