Expression and characterization of catalytic domain of Plasmodium falciparum subtilisin-like protease 3

PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database “PlasmoDB”. The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn334 to Glu769 (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr516 to Glu769 (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes.

79Highlights► PfSUB3 catalytic domain is located in its C-terminal region. ► The region of PfSUB3 spanning from Thr516 to Glu769 possesses serine protease activity. ► PfSUB3 is expressed in late asexual blood stages.