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Abstract:
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.
