Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated

Production of hantavirus Puumala nucleocapsid protein in Saccharomyces cerevisiae for vaccine and diagnostics

Some European hantaviruses can infect humans and cause a serious disease - HFRS. There are no commercially available vaccine and diagnostics against those hantaviruses. The recombinat nucleocapsid protein of hantavirus was demonstrated being a good vaccine candidate and suitable antigen for diagnostics. This study focused on the growth of the recombinant Saccharomyces cerevisiae FH4C strain and respective production of the hantavirus Puumala nucleocapsid protein (N). The recombinant sequence (which encodes N protein) was expressed intracellulary from a S. cerevisiae 2-micron plasmid vector under the control of fused, galactose inducible GAL10-PYK promoter. Different cultivation strategies and media were tested in shake flasks and a 5 L bioreactor, and the respective cell growth, metabolism and nucleocapsid protein production were analyzed. According to requirements for human vaccine production, the traditional medium for yeast cultivation (YEP) can not be used. Therefore the search and screening of an optimal animal origin free medium were performed. The commercial minimal medium for yeast cultivation (YNB) was tested first and it showed a very high specific yield of the recombinant N protein (18.9 mg/g DCW) in batch cultivation, but very low biomass yield (7.5 g/L), therefore the volumetric yield of the recombinant N protein was not very high (142.2 mg/L). When a concentrated minimal medium and fed-batch strategy were used for yeast cultivation, the biomass yield was significantly improved (50 g/L), but the recombinant protein yield decreased drastically. Therefore the commercial (YNB) medium was enriched with plant origin complex components: malt extract and soybean peptone. The best results were obtained from the batch cultivation with malt extract enriched YNB medium: the specific yield of the recombinant N protein was slightly higher (19.4 mg/g DCW) when compared to pure YNB and the biomass yield was higher (11 g/L), therefore the volumetric yield of the recombinant protein was significantly improved (213.6 mg/L). These findings provided the basement of the final and optimal strategy for the recombinant S. cerevisiae cultivation and N protein production

Production of hantavirus Puumala nucleocapsid protein in Saccharomyces cerevisiae for vaccine and diagnostics

The production of hantavirus Puumala nucleocapsid (N) protein for potential applications as a vaccine and for diagnostic purposes was investigated with Saccharomyces cerevisiae as a recombinant host. The N protein gene and the hexahistidine tagged N (h-N) protein gene were expressed intracellular from a 2-μm plasmid vectors under the control of a fused galactose inducible GAL10-PYK promoter. For monitoring the recombinant gene expression, a h-N and a GFP fusion protein was used. Different cultivation strategies and growth media compositions were tested in shake flasks and a 5 l bioreactor. When using defined YNB growth medium, we found the biomass yield to be unsatisfactorily low. Higher concentrated YNB medium, promoted cell growth but showed a pronounced inhibitory effect on heterologous gene expression. This phenomenon could not be attributed to plasmid losses, as we could demonstrate high stability of the vector under the applied cultivation conditions. Supplementation of YNB medium with extracts of plant origin resulted in increased biomass yields with concomitant high expression levels of the recombinant gene. The modified medium was used for fed-batch cultivations where basic metabolic features as well as growth parameters were determined in addition to recombinant gene expression. The maximal volumetric yield of N protein was 316 mg l−1, the respective yield of h-N protein was 284 mg l−1. Our study provides a basis for large-scale production of hantavirus vaccines, which satisfies economic efficiency as well as biosafety regulations for human applications