Functional expression of dipeptidyl peptidase I (Cathepsin C) of the oriental blood fluke Schistosoma japonicum in Trichoplusia ni insect cells.
Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel-nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS-PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH(2)-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues -180 to -175, NH(2)-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH(2)-terminal sequence, LDXNQLY, corresponding to residues -73 to -67 of the proregion peptide and thus were generated by removal of 126 residues from the NH(2)-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37 degrees C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k(cat)/K(m)) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM(-)1 s(-1), respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) High Five cells.
Molecular Parasitology Unit, Royal Brisbane Hospital, Queensland, 4029, Australia.
L
L
Hola-Jamriska
L T
LT
King
J P
JP
Dalton
V H
VH
Mann
J G
JG
Aaskov
P J
PJ
Brindley
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D020789:Cathepsin C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D009036:Moths; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics; D020539:Sequence Analysis, Protein; D018411:Spodoptera
D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D020789:Cathepsin C