RESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Proteínas de Unión al Calcio/genética , Vacunas contra la Malaria/genética , Malaria Falciparum/prevención & control , Glicoproteínas de Membrana/genética , Plasmodium falciparum/química , Proteínas Protozoarias/genética , Tetrahymena thermophila/química , Animales , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Bioensayo , Proteínas de Unión al Calcio/administración & dosificación , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/inmunología , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Inmunogenicidad Vacunal , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Mosquitos Vectores/parasitología , Nanopartículas , Plasmodium falciparum/inmunología , Pliegue de Proteína , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Conejos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Solubilidad , Tetrahymena thermophila/inmunologíaRESUMEN
In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin ß with high nanomolar affinity and specifically inhibits importin α/ß mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin ß and the GTPase Ran. As a selective inhibitor of the importin α/ß import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.