Expression and characterization of the 33kDa and 42kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface-protein-1 (MSP-1(33) and MSP-1(42)) in Escherichia coli

The 42kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface Protein-1 (MSP-1₄₂) is one of the anti-malarial vaccine candidate antigens. In the present study, recombinant MSP-1₄₂ was expressed as a fusion protein in a novel E. coli host. The average yield of the recombinant protein was 24mg/L of bacterial culture. The antigenicity and immunogenicity of the purified protein were evaluated by comparing the results with those obtained from a well characterized recombinant MSP-1₄₂ (Bvp42) expressed in the baculovirus expression system previously described from our laboratory. We observed that the bacterial expressed protein was highly immunogenic in which the immune sera, raised in rabbit and mice, highly cross-reacted with Bvp42. Data from immunoblotting, using two monoclonal antibodies raised against Bvp42 as well as from competitive ELISA studies, suggested that the bacterial expressed protein contains similar B-cell epitopes as that of Bvp42. Subsequent immunoblotting using four disulfide sensitive monoclonal antibodies demonstrated that the recombinant MSP-142 fusion protein is folded in an expected disulfide sensitive conformation similar to that of Bvp42. As two of the monoclonal antibodies have been shown to inhibit parasitic growth in vitro, the reactivity between these antibodies and the protein suggested the presence of inhibitory epitopes on the MSP-1₄₂ fusion protein. Furthermore, cross-reactivity between the two proteins was also observed in T-cell stimulation. By using site direct mutagenesis in modifying the potential enterokinase cleavage site in the MSP-1₄₂ sequence, the unwanted fusion partner could be removed from MSP-1₄₂ by the enzyme without destroying the target protein. Taken together, the similarity in immunological properties between the bacterial expressed MSP-1₄₂ and Bvp42 suggests that the bacterial expression system could be employed to express immunologically active recombinant MSP-142 at elevated levels. This system may be an attractive alternative for producing a protective vaccine for human use at lower cost.; By using similar strategies, the 33kDa processing fragment of P. falciparum (MSP-133) was also expressed in E. coli. The purified protein showed high cross-reactivity in both B-cell and T-cell response with Bvp42 and the bacterial expressed MSP-142 fusion protein. These results indicated that the recombinant MSP-1₃₃ may have similar conformation with the corresponding region in both Bvp42 and the bacterial expressed MSP-142. This protein can be used to study the role of MSP-1 ₃₃ in eliciting protective immunity against P. falciparum in the future.