Falcipain-1, a Plasmodium falciparum cysteine protease with vaccine potential.
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.
Kumar
Amit
A
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Kumar
Krishan
K
Korde
Reshma
R
Puri
Sunil Kumar
SK
Malhotra
Pawan
P
Singh Chauhan
Virander
V
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
D000913:Antibodies, Protozoan; D017780:Malaria Vaccines; D011993:Recombinant Fusion Proteins; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D005780:Gelatin; D003546:Cysteine Endopeptidases; C099607:falcipain