Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum.Accumulated data, including those from nonhuman primate (2, 5) and rodent (1, 3, 16) studies, have indicated that the apical membrane antigen 1 (AMA-1) family of molecules are targets for antibody-mediated protective immune responses. In all Plasmodium species reported to date, with the exception of Plasmodium falciparum (19) and P. reichenowi (13) (two parasites that form a phylogenetic clade distinct from other malaria parasites), AMA-1 is synthesized de novo as a 66-kDa transmembrane protein. The protein contains a predicted Nterminal signal sequence, an ectodomain, a predicted transmembrane region, and a C-terminal cytoplasmic domain. The ectodomain is further divided into three domains defined by disulfide bonds (10). In P. falciparum and P. reichenowi the protein is expressed as an 83-kDa protein, having an N-terminal extension compared to the 66-kDa forms that has been referred to as the prosequence (10). AMA-1 is processed by proteolytic cleavage between the different domains (11). Intraspecies sequence polymorphism due to point mutations (13,15,18,23) reveals clustering of mutations in particular domains of the molecule. Despite this, between species there is considerable conservation of primary and predicted secondary amino acid structures. Evidence to date indicates that protection invoked by AMA-1 is directed at epitopes dependent on the disulfide bonding (1-3, 6, 9, 16) located in the AMA-1 ectodomain. Immunization with reduced AMA-1 fails to induce parasite-inhibitory antibodies (1, 6, 9), and so far only those monoclonal antibodies (MAbs) that recognize reduction-sensitive AMA-1 epitopes have been shown elsewhere to inhibit parasite multiplication in vitro for P. knowlesi (4, 21) and P. falciparum (13,14). This indicates that for an AMA-1 vaccine the correct conformation will be critical.Recombinant expression of P. falciparum AMA-1 (PfAMA...