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

Abstract

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.

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium‐mediated transient gene expression system using Nicotiana benthamiana plants

“…Genetic (e.g., promoter selection and codon optimization), epigenetic (e.g., posttranscriptional gene silencing suppressor) and environmental (e.g., temperature) factors, as well as factors controlling protein stability (physiochemical/biochemical characteristics and the subcellular localizations of recombinant proteins) are the factors (Twyman et al, 2013). Additionally, our recent studies revealed that cultivation conditions, such as the composition of the nutrient solution supplied during plant growth before infiltration with Agrobacterium (agroinfiltration) and desiccation immediately after agroinfiltration, have significant impacts on the recombinant protein content (Fujiuchi et al, 2014(Fujiuchi et al, , 2016b. However, other factors may affect the yield-related quantities.…”

Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system

“…Transient expression in N. benthamiana leaves was performed using transformed A. tumefaciens originating from strain GV3101::pMP90. The bacteria were transformed with a plasmid vector, pNM216 (Fujiuchi et al, ; Matsuda et al, ), derived from pICH26212 (magnICON; Icon Genetics, Halle/Saale, Germany). The T‐DNA present on pNM216 contains cDNA encoding a HA ectodomain derived from influenza A virus strain A/California/07/2009 modified to contain an N‐terminal secretory signal peptide and a C‐terminal endoplasmic reticulum‐retention signal peptide; it also contains cDNAs encoding tobamovirus‐derived RNA‐dependent RNA polymerase and movement protein.…”

“…Before agroinfiltration, 9th and 10th (in Expt I) or 8th and 9th (in Expt II) fully expanded leaves were detached from 35‐day‐old plants. Sixteen (in Expt I) or 28 (in Expt II) detached leaves were submerged in 2.3 L Agrobacterium suspension and vacuum‐infiltrated with the suspension as described in previous studies (Fujiuchi et al, ; Matsuda et al, ). Immediately after agroinfiltration, the detached leaves were removed from the suspension and wiped to eliminate any suspension remaining on the leaf surface.…”

Development of Systems for the Production of Plant-Derived Biopharmaceuticals

“…Plant culture for transient expression is usually divided into two stages, the pre-inoculation and post-inoculation processes, which require different optimal environments. Cultivation conditions, such as the nutrient composition of the culture medium before agro-infiltration [55] and dehydration of plant material after agro-infiltration [70], are key factors. Changing environmental conditions at appropriate times during each stage may be required to obtain a high yield [56]; a high-nitrate nutrient solution during the pre-inoculation state results in high recombinant protein content [55].…”

“…Cultivation conditions, such as the nutrient composition of the culture medium before agro-infiltration [55] and dehydration of plant material after agro-infiltration [70], are key factors. Changing environmental conditions at appropriate times during each stage may be required to obtain a high yield [56]; a high-nitrate nutrient solution during the pre-inoculation state results in high recombinant protein content [55]. Very recently, interest in the surrounding environment has extended beyond research into the plant itself [57,58,71].…”