Pfs48/45 and Pfs230 have been shown to be able to induce transmission blocking immunity, and have thus been considered as TBV candidates. The challenge facing Pfs48/45 and Pfs230 development was to produce the recombinant proteins with the correct conformation. We have been able to express unfolded Pfs48/45 in E. coli and mis-folded/aggregated Pfs48/45 in P. pastoris, but to-date we have been unsuccessful in refolding the protein to its native conformation. The following strategies are being pursued to express the two proteins: ? ? 1. Co-expressing Pfs48/45 with 2 Plasmodium chaperons: Protein Disulphide-Isomerase (PDI); and a heat shock protein DnaJ homologue PfJ2. These chaperons may assist folding and disassembly of the aggregated Pfs48/45 expressed in the Pichia-PDI clone. ? ? 2. Expressing Pfs28/45 in plants as a fusion protein. The project utilizes the technology platform developed by the Center for Molecular and Biotechnology (CMB), Fraunhofer USA. Inc. The Pfs48/45 gene will be inserted into a plant viral vector, which will then be delivered into plant cells for amplification and protein production. The system has been used to produce 30 antigens from various pathogens, including plague, anthrax, influenza, and Trypanosoma brucei. ? ? 3. Expressing Pfs48/45 epitopes as fusions to the coat protein (CP) of Alfalfa mosaic virus (AlMV). This project is also being carried out in collaboration with the CMB of Fruanhofer. The epitopes were selected based on results of epitope mapping of functional monoclonal antibodies against Pfs48/45, and will be inserted into the CP of AlMV expression vector. The vector will be delivered into plant cells, and the epitope-CP fusion protein will be expressed on the surface of the viral particle. This expression system has been used to express >100 peptides of 15-50 amino acids, including peptides from human RSV, HIV, rabies virus, anthrax, and plague.? ? 4. The strategies described in 2) and 3) have also been applied to Pfs230, to express Pfs230 domains in a viral vector, and to express Pfs230 epitopes as fusions to the CP of AlMV.? ? 5. Expressing Pfs230 domains in the Pichia system, and in a wheat germ cell free expression system in collaboration with Ehime Univeristy, Japan.
