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

Abstract

The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

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

“…2B), respectively. The recombinant HA produced in Nicotiana benthamiana leaves agroinfiltrated with the viral vector is generally neither decomposed nor aggregated when retained in endoplasmic reticulum according to immunoblot analysis (Fujiuchi et al, 2016b;Matsuda et al, 2012). The recombinant HA produced in Nicotiana benthamiana leaves agroinfiltrated with the viral vector is generally neither decomposed nor aggregated when retained in endoplasmic reticulum according to immunoblot analysis (Fujiuchi et al, 2016b;Matsuda et al, 2012).…”

“…Leaves at each position, except for position 1-2, were cut into half along the mid vein, and then the HA and total soluble protein (TSP) contents per unit biomass in the leaf halves were quantified as described in a previous study (Fujiuchi et al, 2016b). The recombinant HA produced in Nicotiana benthamiana leaves agroinfiltrated with the viral vector is generally neither decomposed nor aggregated when retained in endoplasmic reticulum according to immunoblot analysis (Fujiuchi et al, 2016b;Matsuda et al, 2012). The distributions of leaf biomass, leaf mass fraction, HA content per unit leaf biomass, and TSP content per unit biomass were obtained.…”

“…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.…”

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20

“…Similarly, A. tumefaciens strain 1D1246 was found to provide high transient expression levels in solanaceous plants without a necrotic response, enabling routine transient expression in solanaceous species. Fujiuchi et al [10] found that residual water from bacterial suspension in the intercellular space of detached leaves could significantly reduce the yield of recombinant protein expression in the syringe agroinfiltration process, and removal of bacterial suspension water in detached leaves after agroinfiltration significantly improved recombinant protein expression.…”

Development of Systems for the Production of Plant-Derived Biopharmaceuticals

“…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].…”