Functional expression of fasciola hepatica cathepsin l1 in saccharomyces cerevisiae
Roche, Leda Dowd, Andrew J. Tort, Jose McGonigle, Sharon McSweeney, Aengus Curley, G. Paul Ryan, Thecla Dalton, John P.
Roche, Leda
Dowd, Andrew J.
Tort, Jose
McGonigle, Sharon
McSweeney, Aengus
Curley, G. Paul
Ryan, Thecla
Dalton, John P.
Publication Date
1997-04-15
Type
Article
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Citation
Roche, Leda; Dowd, Andrew J. Tort, Jose; McGonigle, Sharon; McSweeney, Aengus; Curley, G. Paul; Ryan, Thecla; Dalton, John P. (1997). Functional expression of fasciola hepatica cathepsin l1 in saccharomyces cerevisiae. European Journal of Biochemistry 245 (2), 373-380
Abstract
A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express functionally active enzyme. The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37 degrees C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P-2 position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences.
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Publisher
Wiley-Blackwell
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Attribution-NonCommercial-NoDerivs 3.0 Ireland