FUNCTIONAL EXPRESSION OF FASCIOLA-HEPATICA CATHEPSIN L1 IN SACCHAROMYCES-CEREVISIAE

A cDNA encoding the complete precursor of a Fasciola hepatica cathepsi n L protease was isolated and sequenced. Functionally active enzyme wa s expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sen sitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, whic h lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express functionally active enzyme. The yeast-expr essed enzyme exhibited physicochemical properties in common with the n ative enzyme including, pH optimum for activity, stability at 37 degre es C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have simi lar specificities for substrates with hydrophobic residues in the P-2 position. This is the first report of the functional expression of a c athepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences.