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Infection and Immunity, June 2002, p. 3101-3110, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.3101-3110.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli

Sheetij Dutta,1 P. V. Lalitha,1 Lisa A. Ware,1 Arnoldo Barbosa,1 J. Kathleen Moch,1 Meredith A. Vassell,1 Bader B. Fileta,2 Svetlana Kitov,1 Nelly Kolodny,1 D. Gray Heppner,1 J. David Haynes,1 and David E. Lanar1*

Department of Immunology, Walter Reed Army Institute of Research, Forest Glen Annex, Silver Spring, Maryland 20910,1 Department of Clinical Investigations, Walter Reed Army Medical Center, Washington, D.C. 203072

Received 10 September 2001/ Returned for modification 31 October 2001/ Accepted 12 March 2002

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83Gly to 531Glu) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.

* Corresponding author. Mailing address: Department of Immunology, Walter Reed Army Institute of Research, Forest Glen Annex, Bldg. 503, Silver Spring, MD 20910. Phone: (301) 319-9003. Fax: (202) 318-7594. E-mail: david.lanar{at}na.amedd.army.mil.

Editor: W. A. Petri, Jr.

Infection and Immunity, June 2002, p. 3101-3110, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.3101-3110.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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