title: Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells. Characterization, comparison with the parasite protease, and homology modeling.
abstract: Serine proteases play crucial roles in erythrocyte invasion by merozoites of the malaria parasite. Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is synthesized during maturation of the intraerythrocytic parasite and accumulates in a set of merozoite secretory organelles, suggesting that it may play a role in host cell invasion or post-invasion events. We describe the production, purification, and characterization of recombinant PfSUB-1 and comparison with the authentic protease detectable in parasite extracts. The recombinant protease requires high levels of calcium for optimum activity and has an alkaline pH optimum. Using a series of decapeptide and protein substrates, PfSUB-1 was found to have a relaxed substrate specificity with regard to the P1 position but is unable to efficiently cleave substrates with a P1 leucine residue. Similarly, replacement of a P4 valine with alanine severely reduced cleavage efficiency, whereas its replacement with lysine abolished cleavage. In all respects investigated, the recombinant protease was indistinguishable from parasite-derived enzyme. Three-dimensional homology modeling of the PfSUB-1 catalytic domain based on an alignment with closely related bacterial subtilisins and an orthologue from the rodent malaria Plasmodium yoelii suggests that the protease has at least three potential calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine within the enzyme S1 pocket. A predicted highly polar S1 pocket and a hydrophobic S4 subsite are in broad agreement with the experimentally determined substrate specificity.
authors.affiliation: Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Chrislaine
authors.initials: C
authors.lastname: Withers-Martinez
authors.forename: José W
authors.initials: JW
authors.lastname: Saldanha
authors.forename: Barry
authors.initials: B
authors.lastname: Ely
authors.forename: Fiona
authors.initials: F
authors.lastname: Hackett
authors.forename: Tony
authors.initials: T
authors.lastname: O'Connor
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Blackman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017931:DNA Primers; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; C403176:subtilisin-like protease 1, Plasmodium falciparum