title: Falcipain-1, a Plasmodium falciparum cysteine protease with vaccine potential.
abstract: Cysteine proteases (falcipains) of Plasmodium falciparum are potential targets for antimalarial chemotherapy, since they have been shown to be involved in important cellular functions such as hemoglobin degradation and invasion/rupture of red blood cells during parasite life cycle. The role of falcipain-1 at the asexual blood stages of the parasite still remains uncertain. This is mainly due to a lack of methods to prepare this protein in an active form. In order to obtain biologically active falcipain-1, a number of falcipain-1 constructs were designed and a systematic assessment of the refolding conditions was done. We describe here the expression, purification, and characterization of a falcipain-1 construct encoding mature falcipain-1 and 35 amino acids from the C-terminal of the pro domain. Recombinant falcipain-1 was overexpressed in the form of inclusion bodies, solubilized, and purified by Ni(2+)-nitrilotriacetic acid affinity chromatography under denaturing conditions. A systemic approach was then followed to optimize refolding parameters. An optimum refolding condition was obtained, and the yield of the purified refolded falcipain-1 was approximately 1 mg/liter. Activity of the protein was analyzed by fluorometric and gelatin degradation assays. Immunolocalization studies using anti-falcipain-1 sera revealed a distinct staining at the apical end of the P. falciparum merozoites. Previous studies using falcipain-1-specific inhibitors have suggested a role of falcipain-1 in merozoite invasion. Based on its localization and its role in invasion, we analyzed the immunogenicity of falcipain-1 in mice, followed by heterologous challenge with Plasmodium yoelii sporozoites. Our results suggest a possible role of falcipain-1 in merozoite invasion.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.forename: Krishan
authors.initials: K
authors.lastname: Kumar
authors.forename: Reshma
authors.initials: R
authors.lastname: Korde
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Puri
authors.forename: Pawan
authors.initials: P
authors.lastname: Malhotra
authors.forename: Virander
authors.initials: V
authors.lastname: Singh Chauhan
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D005470:Fluorometry; D005780:Gelatin / Q000378:metabolism; D007150:Immunohistochemistry; D008288:Malaria / Q000517:prevention & control; D017780:Malaria Vaccines / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence; D018512:Parasitemia; D010963:Plasmodium falciparum / Q000737:chemistry / Q000201:enzymology* / Q000276:immunology*; D010967:Plasmodium yoelii / Q000276:immunology; D017510:Protein Folding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D022223:Vaccines, Subunit / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D017780:Malaria Vaccines; D011993:Recombinant Fusion Proteins; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D005780:Gelatin; D003546:Cysteine Endopeptidases; C099607:falcipain