Functional expression of fasciola hepatica cathepsin l1 in saccharomyces cerevisiae
Dowd, Andrew J.
Curley, G. Paul
Dalton, John P.
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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
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.