RESUMO
A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25â¯nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Proteínas de Ligação ao Cálcio/genética , Vacinas Antimaláricas/genética , Malária Falciparum/prevenção & controle , Glicoproteínas de Membrana/genética , Plasmodium falciparum/química , Proteínas de Protozoários/genética , Tetrahymena thermophila/química , Animais , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Bioensaio , Proteínas de Ligação ao Cálcio/administração & dosagem , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imunogenicidade da Vacina , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Mosquitos Vetores/parasitologia , Nanopartículas , Plasmodium falciparum/imunologia , Dobramento de Proteína , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Coelhos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Solubilidade , Tetrahymena thermophila/imunologiaRESUMO
We report here the first purification of a P(1B) type ATPase, a group of transporters that occurs in bacteria, plants and animals incl. humans, from a eukaryotic organism in native state. TcHMA4 is a P(1B) type ATPase that is highly expressed in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens and contains a C-terminal 9-histidine repeat. After isolation from roots, we purified TcHMA4 protein via metal affinity chromatography. The purified protein exhibited Cd- and Zn-activated ATPase activity after reconstitution into lipid vesicles, showing that it was in its native state. Gels of crude root extract and of the purified protein revealed TcHMA4-specific bands of about 50 and 60kDa, respectively, while the TcHMA4 mRNA predicts a single protein with a size of 128kDa. This indicates the occurrence of post-translational processing; the properties of the two bands were characterised by their activity and binding properties.