Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli.

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 25 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.

The effect of the aqueous extract of the roots of Asparagus racemosus on hepatocarcinogenesis initiated by diethylnitrosamine.

Histopathological studies of the hepatic tissues of Wistar rats treated with diethylnitrosamine (DEN) (200 mg/kg b wt, i.p.) once a week for 2 weeks, followed by treatment with DDT, a tumor promoter (0.05% in diet) for 2 weeks and kept under observation for another 18 weeks, demonstrated the development of malignancy. Pretreatment of Wistar rats with the aqueous extract of the roots of Asparagus racemosus prevented the incidence of hepatocarcinogenesis. Immunohistochemical staining of the hepatic tissues of rats treated with DEN showed the presence of p53+ foci (clusters of cells expressing the mutated p53 protein), whereas an absence of p53+ foci was observed in Wistar rats pretreated with the aqueous extract of the roots of Asparagus racemosus. The microsections of the hepatic tissue of rats treated with DEN followed by treatment with the aqueous extract of Asparagus racemosus showed an absence of p53+ foci. The results of the biochemical determinations also show that pretreatment of Wistar rats with the aqueous extract of Asparagus racemosus leads to the amelioration of oxidative stress and hepatotoxicity brought about by treatment with DEN. These results prove that the aqueous extract of the roots of Asparagus racemosus has the potential to act as an effective formulation to prevent hepatocarcinogenesis induced by treatment with DEN.