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Reducing Batch-to-Batch Variability of Agrobacterium-Mediated Transient Protein Expression in Plant Tissue Culture

Jeffrey S. Larsen and Wayne R. Curtis. Chemical Engineering, The Pennsylvania State University, 133 Fenske Laboratory, University Park, PA 16802

Batch-to-batch variations in protein expression levels pose an intractable and often unreported technical hurdle in the development of rapid plant-based heterologous protein expression platforms. Our group is developing protein expression in non-transgenic plant tissue culture which is grown and then co-cultured with an Agrobacterium tumefaciens auxotroph to rapidly deliver heterologous DNA to the nucleus of the plant cells. Transient expression of this extra-chromosomal DNA results in a temporal peak in heterologous protein expression in three to five days. We have successfully demonstrated this technology at the 50-L pilot scale. However, even after systematically improving reproducibility via rigor in culture maintenance and synchronization, the concentration of the reporter gene product β-glucuronidase has been observed to vary by orders of magnitude from one experiment to the next. Similar large variations have been observed in plant leaves infiltrated with Agrobacterium.

Achieving reproducible product titers is crucial for developing this protein expression technology for applications ranging from high-throughput screening to commercial-scale production of therapeutic proteins. For large-scale production, predictable and high-level productivity is needed to minimize capital investment as well as develop downstream processing and purification.

Plant tissue culture-based transient expression provides an ideal platform for controlling variability once the required environmental conditions are identified. Physical and physiological sources of batch-to-batch variation in high-level transient protein expression will be discussed in detail. Some of the important parameters that have been identified and studied as potential contributors to this variation include the organic and inorganic nutrient status of the culture, culture maintenance schedule, gas exchange in shake-flasks, incubator temperature control, and the timing of the nutrient-controlled 'synchronization by starvation' relative to the co-culture of the plant tissues with A. tumefaciens.