Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae.
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.
Dowd
A J
AJ
School of Biological Sciences, Dublin City University, Glasnevin, Ireland.
Tort
J
J
Roche
L
L
Ryan
T
T
Dalton
J P
JP
D000818:Animals; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003546:Cysteine Endopeptidases; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D018509:DNA, Helminth / Q000235:genetics* / Q000302:isolation & purification; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000235:genetics; D005210:Fasciola hepatica / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D005340:Fibrinogen / Q000378:metabolism; D015870:Gene Expression; D017238:Genes, Helminth; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D005340:Fibrinogen; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C055808:preprocathepsin L; D056668:Cathepsin L