Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae.
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 funtionally 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 P2 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.
Roche
L
L
School of Biological Sciences, Dublin City University, Republic of Ireland.
Dowd
A J
AJ
Tort
J
J
McGonigle
S
S
MacSweeney
A
A
Curley
G P
GP
Ryan
T
T
Dalton
J P
JP
McSweeney
A
A
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000302:isolation & purification; D018509:DNA, Helminth / Q000302:isolation & purification / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics; D005210:Fasciola hepatica / Q000201:enzymology* / Q000235:genetics; D005780:Gelatin / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D018509:DNA, Helminth; D004274:DNA, Recombinant; D004792:Enzyme Precursors; D007074:Immunoglobulin G; D005780:Gelatin; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C055808:preprocathepsin L; D056668:Cathepsin L