Vaccine Candidate Msp-1 From Plasmodium Falciparum: A Redesigned 4917 Bp Polynucleotide Enables Synthesis And Isolation Of Full-Length Protein From Escherichia Coli And Mammalian Cells

The Plasmodium falciparum malaria parasite is the causative agent of malaria tropica, Merozoites, one of the extracellular developmental stages of this parasite, expose at their surface the merozoite surface protein-1 complex (MSP-1), which results from the proteolytic processing of a 190-200 kDa precursor. MSP-1 is highly immunogenic in humans and numerous studies suggest that this protein is an effective target for a protective immune response, Although its function is unknown, there are indications that it may play a role during invasion of erythrocytes by merozoites, The parasite-derived msp-1 gene, which is similar to 5000 bp long, contains 74% AT, This high AT content has prevented stable cloning of the full-size gene in Escherichia coli and consequently its expression in heterologous systems. Here, we describe the synthesis of a 4917 bp gene encoding MSP-1 from the FCB-1 strain of P. falciparum adjusted for human codon preferences. The synthetic msp-1 gene (55% AT) was cloned, maintained and expressed in its entirety in E.coli as well as in CHO and HeLa cells. The purified protein is soluble and appears to possess native conformation because it reacts with a panel of mAbs specific for conformational epitopes, The strategy we used for synthesizing the full-length msp-1 gene was to assemble it from DNA fragments encoding all of the major proteolytic fragments normally generated at the parasite's surface, Thus, after subcloning we also obtained each of these MSP-1 processing products as hexahistidine fusion proteins in E.coli and isolated them by affinity chromatography on Ni2+ agarose, The availability of defined preparations of MSP-1 and its major processing products open up new possibilities for in-depth studies at the structural and functional level of this important protein, including the exploration of MSP-1-based experimental vaccines.