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

Protein Expr Purif. 2019 Jan:153:7-17. doi: 10.1016/j.pep.2018.08.001. Epub 2018 Aug 3.


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.

Keywords: Bacterial protein expression; Malaria; Particle-based vaccine; Plasmodium; Solubility tag; Tetrahymena.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Protozoan / biosynthesis*
  • Antigens, Protozoan / administration & dosage
  • Antigens, Protozoan / chemistry
  • Antigens, Protozoan / genetics
  • Antigens, Protozoan / immunology
  • Biological Assay
  • Calcium-Binding Proteins / administration & dosage
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / genetics*
  • Calcium-Binding Proteins / immunology
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Immunogenicity, Vaccine
  • Malaria Vaccines / administration & dosage
  • Malaria Vaccines / genetics*
  • Malaria, Falciparum / immunology
  • Malaria, Falciparum / parasitology
  • Malaria, Falciparum / prevention & control*
  • Membrane Glycoproteins / administration & dosage
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics*
  • Membrane Glycoproteins / immunology
  • Mosquito Vectors / parasitology
  • Nanoparticles
  • Plasmodium falciparum / chemistry*
  • Plasmodium falciparum / immunology
  • Protein Folding
  • Protozoan Proteins / administration & dosage
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics*
  • Protozoan Proteins / immunology
  • Rabbits
  • Recombinant Fusion Proteins / administration & dosage
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Solubility
  • Tetrahymena thermophila / chemistry*
  • Tetrahymena thermophila / immunology


  • Antibodies, Protozoan
  • Antigens, Protozoan
  • Calcium-Binding Proteins
  • GRL1 protein, Tetrahymena thermophila
  • Malaria Vaccines
  • Membrane Glycoproteins
  • Pfs25 protein, Plasmodium falciparum
  • Protozoan Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • pfs48-45 protein, Plasmodium falciparum