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

Abstract

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.

Tetrahymena thermophila Granule Lattice Protein 3 Improves Solubility of Sexual Stage Malaria Antigens Expressed in Escherichia coli

“…While the majority of this protein appeared in the insoluble fraction, bands of the same size were also present in the soluble fractions. As noted previously (Agrawal et al, 2019; Chilcoat et al, 1996), Grl1p migrates anomalously on SDS-PAGE which likely accounts for the difference in the predicted molecular mass of the Pfs25-Grl1p chimera (65kDa) compared with the size determined by gel electrophoresis (∼90 kDa) (Figure 1). Varying the IPTG concentrations between 50-500 mM had only a small effect on overall expression (Figure 1), while induction at 30°C resulted in significantly lower protein expression than at 16°C.…”

“…Indeed, by combining expression in SHuffle with co-expression of selected chaperones and/or the use of solubility tags, further improvements in solubility can be achieved (Bessette et al, 1999; Jurado et al, 2002; Yuan et al, 2004). Notably, this combined approach was effective in generating soluble forms of the malaria transmission-blocking vaccine, Pfs25, as a chimera with the granule-lattice protein, Grl1p, from Tetrahymena thermophila (Agrawal et al, 2019). In the absence of tags, soluble Pfs25 was barely detectable even in the SHuffle strain, while Pfs48/45 appeared to be completely insoluble (Agrawal et al, 2019).…”

“…Digested DNA was run on agarose gel (1%) electrophoresis and a fragment corresponding to the Grl3p cDNA was purified by gel extraction. The fragment was then ligated to the plasmids containing either Pfs25 or Pfs48/45 as previously described (Agrawal et al, 2019).…”

“…Frozen cell pellets were thawed on ice and resuspended in 20mM Tris, 300mM NaCl, 5% glycerol, 1% Tween-80, and 15mM imidazole (pH 8.0) containing protease inhibitors (Roche cOmplete EDTA-free) as previously described (Agrawal et al, 2019). Cell suspensions were lysed by sonication (10 cycles of 10 sec on, 10 sec off at 80% power) (Qsonica Sonicator Q125, Fisher Scientific).…”

“…Recently, we showed that granule lattice protein 1 (Grl1p) of the free-living ciliate Tetrahymena thermophila substantialy improves protein solubility when expressed as a C-terminal fusion with the transmission-blocking vaccine candidate, Pfs25, in E. coli strains that co-express bacterial chaperone proteins (Agrawal et al, 2019). Grl1p is one of a small family of acidic proteins that populate dense core secretory granules of T. thermophila and that can self-assemble into nanometer-sized particles in vivo and in vitro (Cowan et al, 2005).…”