Scale‐Up of Agrobacterium‐Mediated Transient Protein Expression in Bioreactor‐Grown Nicotiana glutinosa Plant Cell Suspension Culture

Abstract

The reporter gene -glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through coculture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to transient expression including the timing of Agrobacterium inoculation relative to the plant cell growth phase, plant tissue culture hormonal triggers and plant cell cycle synchronization. The co-culture process was simplified to facilitate implementation in a pilot-scale bioreactor. At the shake flask scale it was determined that elevated concentrations of oxygen in the headspace were detrimental to transient expression levels and the addition of acetosyringone to the co-culture had a negligible effect. The bacterial preparation process was also streamlined, permitting the direct transfer of the Agrobacterium culture from a bench-scale fermentor to the pilot-scale plant cell culture bioreactor. Increasing expression levels and overcoming batch-to-batch variability despite extensive procedure systemization remain the major technical hurdles.

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

“…Relative to cell suspensions, mature leaves may provide a more natural environment for the attachment of A. tumefaciens and subsequent transfer of T-DNA. Yet, the highest yields for transient expression have been achieved in heterotrophic plant cell suspensions undergoing rapid cell division, whereas non-dividing but metabolically active cells in fully expanded leaves tend to work best in planta [50,51]. Consequently, differences in physiological state other than cell cycle status must play an important role in mediating transient protein expression of ectopic DNA.…”

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…Several systems have been developed for transient expression of foreign proteins in plant suspension and hairy root cultures based on viral vectors or agrobacterial co-culture [29,39,[50][51][52]. This approach restricts transformation and protein expression to only certain stages of cell culture, thereby avoiding the reductions in expression levels that occur during multiple cell divisions due to mutations or genetic re-arrangements.…”

“…Because chemical agents are applied easily to plant cultures by direct addition to the medium, a wide variety of inducible promoters activated by compounds such as ethanol, estradiol, salicylic acid, dexamethasone and tetracycline [61] are also suitable for in vitro application. In principle, transient transformation and expression systems [29,39,[50][51][52] coupled with knowledge of the timecourse of protease production and/or secretion could also be used to allow higher levels of foreign protein to accumulate in the absence of major protease effects.…”

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

“…GUS activity was quantified by a fluorometric assay with a 4-methylumbeliferyl B–D-glucuronide (MUG; Sigma) substrate [30]. The value was reported as the mean of the ratio of the molar rate of formation of 4-methyl-7-hexacoumarin (MU) to the total soluble protein.…”