Blocking endocytotic mechanisms to improve heterologous protein titers in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a useful platform for protein production of biopharmaceuticals and industrial enzymes. To date, substantial effort has focused on alleviating several bottlenecks in expression and the secretory pathway. Recently, it has been shown that highly active endocytosis could decrease the overall protein titer in the supernatant. In this study, we block endocytosis and trafficking to the vacuole using a modified TEV Protease-Mediated Induction of Protein Instability (mTIPI) system to disrupt the endocytotic and vacuolar complexes. We report that conditional knock-down of endocytosis gene Rvs161 improved the concentration of α-amylase in supernatant of S. cerevisiae cultures by 63.7% compared to controls. By adaptive evolution, we obtained knock-down mutants in Rvs161 and End3 genes with 2-fold and 3-fold α-amylase concentrations compared to controls that were not evolved. Our study demonstrates that genetic blocking of endocytotic mechanisms can improve heterologous protein production in S. cerevisiae. This result is likely generalizable to other eukaryotic secretion hosts.

Immunogenicity and protective efficacy of yeast extracts containing rotavirus-like particles: a potential veterinary vaccine.

Rotavirus is the most common cause of severe diarrhea in many animal species of economic interest. A simple, safe and cost-effective vaccine is required for the control and prevention of rotavirus in animals. In this study, we evaluated the use of Saccharomyces cerevisiae extracts containing rotavirus-like particles (RLP) as a vaccine candidate in an adult mice model. Two doses of 1mg of yeast extract containing rotavirus proteins (between 0.3 and 3 µg) resulted in an immunological response capable of reducing the replication of rotavirus after infection. Viral shedding in all mice groups diminished in comparison with the control group when challenged with 100 50% diarrhea doses (DD50) of murine rotavirus strain EDIM. Interestingly, when immunizing intranasally protection against rotavirus infection was observed even when no increase in rotavirus-specific antibody titers was evident, suggesting that cellular responses were responsible of protection. Our results indicate that raw yeast extracts containing rotavirus proteins and RLP are a simple, cost-effective alternative for veterinary vaccines against rotavirus.

The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice.

Viral protein assemblies have shown to be superior immunogens used in commercial vaccines. However, little is known about the effect of protein assembly structure in immunogenicity and the protection conferred by a vaccine. In this work, rotavirus VP6, a polymorphic protein that assembles into nanotubes, icosahedra (dlRLP) or trimers was used to compare the immune response elicited by three different assemblies. VP6 is the most antigenic and abundant rotavirus structural protein. It has been demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it a vaccine candidate. Groups of mice were immunized with either nanotubes, dlRLP or trimers and the humoral response (IgG and IgA titers) was measured. Immunized mice were challenged with EDIM rotavirus and protection against rotavirus infection, measured as viral shedding, was evaluated. Immunization with nanotubes resulted in the highest IgG titers, followed by immunization with dlRLP. While immunization with one dose of nanotubes was sufficient to reduce viral shedding by 70%, two doses of dlRLP or trimers were required to obtain a similar protection. The results show that the type of assembly of VP6 results in different humoral responses and protection efficacies against challenge with live virus. This information is important for the design of recombinant vaccines in general.

Effect of metal catalyzed oxidation in recombinant viral protein assemblies.

BACKGROUND: Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein. RESULTS: The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased. CONCLUSIONS: Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of oxidation in protein assembly is studied, evidencing that oxidation should be minimized during the production process if VP6 nanotubes are required.

Virus-like particles: the future of microbial factories and cell-free systems as platforms for vaccine development.

Vaccines based on virus-like particles have proved their success in human health. More than 25 years after the approval of the first vaccine based on this technology, the substantial efforts to expand the range of applications and target diseases are beginning to bear fruit. The incursion of high-throughput screening technologies, combined with new developments in protein engineering and chemical coupling, have accelerated the development of systems capable of producing macrostructures useful for vaccinology, gene delivery, immunotherapy and bionanotechnology. This review summarizes the most recent developments in microbial cell factories and cell-free systems for virus-like particle production and discusses the future impact of this technology in human and animal health.

Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae.

BACKGROUND: Virus-like particles (VLP) have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP) in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. RESULTS: The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp) or episomal (YEp), did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. CONCLUSIONS: We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts could lead to the development of new vaccine candidates with reduced restrictions by regulatory agencies, using the successful experience with other yeast-based VLP vaccines commercialized worldwide.

Genotypification of bovine group A rotavirus in México.

Bovine scours, frequently provoked by rotavirus infection, causes significant economic losses. Nevertheless, no information exists about the bovine rotavirus genotypes present in Mexico. This information is necessary for designing efficient vaccines. In this work, 128 samples from diarrheic calves were collected between 2005 and 2006 in 26 dairy and/or beef cattle herds located in 10 regions of Mexico, and analyzed for the presence of group A rotavirus. G and P genotypes were determined by PCR in rotavirus-positive samples (12/128). Three different genotype combinations were found, G10, P[11]; G6, P[5]; and G10, P[5]; in 67, 25 and 8% of the positive samples, respectively. Some rotavirus-positive animals had been vaccinated with an inactivated rotavirus strain of a different genotype.