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Viral vector-mediated transient expression enables rapid biopharmaceutical protein production using whole plants. In recent years, plant growth conditions have received close attention as factors influencing the content of recombinant protein. In particular, because nitrogen is an important resource used in protein synthesis, nitrogen supplied to the root zone may influence accumulation of recombinant proteins. We tested whether fertilization of Nicotiana benthamiana with high-nitrate nutrient solution during plant growth prior to Agrobacterium-mediated delivery (agroinfiltration) of a tobamoviral vector resulted in high recombinant influenza hemagglutinin (HA) content per unit of leaf fresh weight and/or per plant. Nutrient solution containing 60 mM nitrate yielded 40% higher HA content per unit of leaf fresh weight, and comparable HA content per plant, relative to nutrient solution containing 12 or 36 mM nitrate. There were positive correlations among HA, total soluble protein (TSP), and soluble reduced-nitrogen content, suggesting that fertilization with the high-nitrate nutrient solution increased soluble reduced nitrogen and subsequently provided high TSP and HA. Although the increased HA content per unit of leaf fresh weight was offset by reduced leaf fresh weight on a per plant basis, nitrate-enriched fertilization represents an easily applicable technique to obtain a given amount of HA from smaller leaf biomass, thereby potentially reducing downstream processing costs for HA vaccine production using viral vector-mediated transient expression systems.

plant biotechnology

Effect of nitrate concentration in nutrient solution on hemagglutinin content of Nicotiana benthamiana leaves in a viral vector-mediated transient gene expression system