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 purifled 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. NH₂-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues -180 to -175, NH₂-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH₂-terminal sequence, LDXNQLY, corresponding to residues -73 to -67 of the proregion peptide and thus were generated by removal of 126 residues from the NH₂-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37°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 ~2 x 10⁹ High Five cells. (C) 2000 Academic Press.