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Infection and Immunity, April 2007, p. 2026-2034, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01533-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Falcipain-1, a Plasmodium falciparum Cysteine Protease with Vaccine Potential{triangledown}

Amit Kumar,{dagger} Krishan Kumar, Reshma Korde, Sunil Kumar Puri,{ddagger} Pawan Malhotra,* and Virander Singh Chauhan*

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

Received 22 September 2006/ Returned for modification 24 October 2006/ Accepted 21 December 2006

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 Ni2+-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 ~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.

* Corresponding author. Mailing address: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Phone: 91 11 26189358. Fax: 91 11 26162316. E-mail for Virander Singh Chauhan: virander{at}icgeb.res.in. E-mail for Pawan Malhotra: pawanm{at}icgeb.res.in.

{triangledown} Published ahead of print on 22 January 2007.

Editor: W. A. Petri, Jr.

{dagger} Present address: 128 Polk Hall, NCSU, Raleigh, NC 27695.

{ddagger} Present address: CDRI, Chattar Manzil Palace, Lucknow, UP 226001, India.

Infection and Immunity, April 2007, p. 2026-2034, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01533-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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