Oral immunization against foot-and-mouth disease virus using recombinant Saccharomyces cerevisiae with the improved expression of the codon-optimized VP1 fusion protein.

VP1, a major immunogenic protein of foot-and-mouth disease virus (FMDV), facilitates viral attachment and entry into host cells. VP1 possesses critical epitope sequences responsible for inducing neutralizing antibodies but its expression using Saccharomyces cerevisiae has been hampered despite evidence that the presence of VP1 does not negatively impact the yeast's biology. In this study, we fused proteins to enhance VP1 expression using S. cerevisiae. Among short P1 chimeras containing VP1 including VP3-VP1 and VP2-VP1, VP3-VP1 fusion proteins showed higher expression levels than VP2-VP1. We subsequently designed new fusion proteins, of which 20 amino acids of N-terminal VP3 fused with VP1-Co1 (referred to 20aaVP3-VP1-Co1) showed the highest expression level. Lowering the culture temperature from 30 °C to 20 °C further enhanced fusion protein production. The highest expression level of 20aaVP3-VP1-Co1 was estimated to be 7.7 mg/L, which is comparable to other heterologous proteins produced using our S. cerevisiae expression system. Oral administration of the cell expressing 20aaVP3-VP1-Co1 induced VP1-specific IgG and IgA responses in mice. The S. cerevisiae-expressed 20aaVP3-VP1-Co1 fusion protein induced a significant immune response to the FMDV structural epitope protein, which opens the possibility of an oral FMDV vaccine.

Expression of virus-like particles (VLPs) of foot-and-mouth disease virus (FMDV) using Saccharomyces cerevisiae.

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational "ribosomal skipping" sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a "ribosomal skipping" signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. KEY POINTS: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle.

Improving expression and assembly of difficult-to-express heterologous proteins in Saccharomyces cerevisiae by culturing at a sub-physiological temperature.

BACKGROUND: Escherichia coli heat labile toxin B subunit (LTB) is one of the most popular oral vaccine adjuvants and intestine adsorption enhancers. It is often expressed as a fusion partner with target antigens to enhance their immunogenicity as well as gut absorbability. However, high expression levels of a fusion protein are critical to the outcome of immunization experiments and the success of subsequent vaccine development efforts. In order to improve the expression and functional assembly of LTB-fusion proteins using Saccharomyces cerevisiae, we compared their expression under culture conditions at a sub-physiological temperature 20 °C with their expression under a standard 30 °C. RESULTS: The assembled expression of LTB-EDIII2 (LTB fused to the envelope domain III (EDIII) of Dengue virus serotype 2), which was expressed at the level of 20 µg/L in our previous study, was higher when the expression temperature was 20 °C as opposed to 30 °C. We also tested whether the expression and functional assembly of a difficult-to-express LTB fusion protein could be increased. The assembled expression of the difficult-to-express LTB-VP1 fusion protein (LTB fused to VP1 antigen of Foot-and-Mouth Disease Virus) dramatically increased, although the total amount of expressed protein was still lower than that of LTB-EDIII2. Slight but significant increase in the expression of well-known reporter protein eGFP, which has previously been shown to be increased by cultivation at 20 °C, was also observed in our expression system. As no significant changes in corresponding transcripts levels and cell growth were observed between 20 °C and 30 °C, we infer that translation and post-translational assembly are responsible for these enhancements. CONCLUSIONS: The effects of lowering the expression temperature from 30 °C to 20 °C on protein expression and folding levels in S. cerevisiae, using several proteins as models, are reported. When heterologous proteins are expressed at 20 °C, a greater amount of (specially, more assembled) functional proteins accumulated than at 30 °C. Although further studies are required to understand the molecular mechanisms, our results suggest that lowering the expression temperature is a convenient strategy for improving the expression of relatively complexly structured and difficult-to-express proteins in S. cerevisiae.