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

Jan 2019, ScienceDirect

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. falciparumproteins 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. coliSHuffle 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 proteinsand 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.

A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

May-Jun 2018, MAbs

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.

An Alternative Platform for Rapid Production of Effective Subunit Vaccines

Oct 2010, BioPharm International

The freshwater ciliate, Tetrahymena thermophila, has recently emerged as a novel manufacturing platform for recombinant subunit vaccines. It combines ease of growth with facile genetics in a complex unicellular eukaryote that can be grown rapidly in inexpensive media on an industrial scale. T. thermophila devotes a large part of its metabolism to membrane protein production, and can release proteins to the extracellular space by constitutive and stimulus-dependent pathways of secretion. In this article, we show high-level expression of correctly folded parasite and viral proteins in a Tetrahymena system and provide direct evidence that regulated secretion can be harnessed as an effective pathway for producing influenza hemagglutinin (HA). HA can be targeted to dense core granules in vivo and be recovered following stimulus-dependent secretion in association with a proteinaceous gel termed PRISM. PRISM offers a convenient matrix for protein purification, but at the same time, has intrinsic properties with the potential to induce potent immune responses to co-administered antigens.

Constitutive Secretion in Tetrahymena thermophila

May 2010, Eukaryotic Cell

The growth, survival, and life cycle progression of the freshwater ciliated protozoan Tetrahymena thermophila are responsive to protein signals thought to be released by constitutive secretion. In addition to providing insights about ciliate communication, studies of constitutive secretion are of interest for evaluating the utility of T. thermophila as a platform for the expression of secreted protein therapeutics. For these reasons, we undertook an unbiased investigation of T. thermophila secreted proteins using wild-type and secretion mutant strains. Extensive tandem mass spectrometry analyses of secretome samples were performed. We identified a total of 207 secretome proteins, most of which were not detected in a set of abundant whole-cell protein identifications. Numerous proteases and other hydrolases were secreted from cells grown in rich medium but not cells transferred to a nutrient starvation condition. On the other hand, we detected the starvation-enhanced secretion of a small number of cytosolic proteins, suggestive of an exosome-like pathway in T. thermophila. Subsets of proteins from the T. thermophila regulated secretion pathway were detected with differential representation across strains and culture conditions. Finally, many secretome proteins had a predicted N-terminal signal sequence but no other annotated characteristic or functional classification. Our work provides the first comprehensive analysis of secreted proteins in T. thermophila and establishes the groundwork for future studies of constitutive protein secretion biology and biotechnology in ciliates.