title: 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.
authors.affiliation: Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657, Japan. naofujiuchi@gmail.com.
authors.forename: Naomichi
authors.initials: N
authors.lastname: Fujiuchi
authors.affiliation: Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Matsuda
authors.affiliation: Owensboro Cancer Research Program, James Graham Brown Cancer Center, University of Louisville School of Medicine, Owensboro, Kentucky.|Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Matoba
authors.affiliation: Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Fujiwara
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D005110:Extracellular Space / Q000737:chemistry*; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism*; D018526:Plant Transpiration; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014867:Water / Q000378:metabolism*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; D014867:Water
keywords: influenza vaccine; molecular farming; plant-made pharmaceutical (PMP); tobacco; viral vector