Biotechnology and Bioengineering · 2011 · 33 citations · 39 references
AgricultureAgricultural BiotechnologyBiotechnologyPlant BiotechnologyProtein EngineeringTransgenic CropsAgrobacterium-mediated TransformationCell Culture TechnologyProtein Expression EngineeringRecombinant Protein ExpressionRecombinant Protein ProductionTransgenic Organism ProductionAgrobacterium-mediated Transformation ConstructsCell Culture ProductionCloningDirectional CloningPlant-based ProductionPlant-based Protein ExpressionRecombinant Fusion ProteinsScalable Cell Culture Systems
To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.
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Ajay Kohli, Mark J. Leech, Phillipe Vain et al. · Proceedings of the National Academy of Sciences · 1998 · 262 citations