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-microm 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.
Antoniukas
L
L
Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany. linasa@ibt.lt
Grammel
H
H
Reichl
U
U
D018533:Biomass; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000096:biosynthesis*; D029262:Puumala virus / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D022223:Vaccines, Subunit / Q000096:biosynthesis; D014765:Viral Vaccines / Q000096:biosynthesis*
D003470:Culture Media; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; D014765:Viral Vaccines; D005947:Glucose