Table 2. Proteins Expressed Using Tobacco Transformation Chloroplast Technology4, 9-14
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A fertilizer regime is used to encourage the regeneration of multiple secondary shoots every four weeks, providing a maximum
of four harvests during the growing season. Three harvests during a single growing season generated biomass up to 297,000
lbs/acre.
Biomass Production Yield
Average yields for a growing season of high-density tobacco fields are approximately 55 tons/acre or 110,000 lbs/acre. Averaged
total soluble protein (TSP) per unit biomass can be used to predict the total crude target-protein yield on an acreage basis.
A predictive protein-yield graph based on a range of expression levels and acreage yields is shown in Figure 4.
Published reports describing protein expression levels for a number of protein classes have identified ranges between 2 and
40% total soluble protein (TSP), depending on the specific protein.4,9,10,12-14 If one assumes a nominal expression level of 8% TSP and a seasonal crop of 55 tons/acre, the crude protein yield specified
in Figure 4 is approximately 97 lbs/acre. This is an extremely large target-protein-to-biomass ratio, which translates into
several advantages during processing. A 20% TSP-producing cultivar can generate up to 265 pounds of crude recombinant protein
per acre.
Figure 3. Modular Components of the Manufacturing Process Using Green Tobacco Biomass
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A high target-protein-to-biomass ratio allows for a reduction in the size of upstream processing equipment. For example, compare
processing tobacco with a 1:100 target protein-to-biomass ratio versus a 1:1000 ratio. Within equal time periods, we can expect
a tenfold reduction in the size of the equipment used in upstream processing, resulting in lower capital and overall costs.
The initial concentration of target protein also impacts downstream processing in terms of volume reduction steps and ratios
of transgene protein to host and environmental contaminating molecules — another saving.
Pre-Extraction
Green biomass may need to be manipulated to prepare the material for extraction. For green biomass, pre-extraction processing
generally refers to comminution or pulverizing the material into specific-sized particles that will enhance the subsequent
extraction process. Comminution is generally required when increased surface area or reduced diffusion distances are required.
Typical comminution methods include cutting, grinding, and milling.14
Extraction
Extraction is based on two complementary processes running parallel to each other.
- Rinsing target protein out of disintegrated chloroplasts and plant cells
- Dissolution of target protein from plant material by diffusion (the resulting extraction solution is called the miscella)
Greenhouse production of transgenic tobacco transplants using a traditional “float tray” system
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Extraction processes can be further divided into two major groups.
- Processes that result in a concentration equilibrium between solute and target protein
- Processes in which the target protein is extracted exhaustively.17
There are many methods of extraction including ultrasonic, electromagnetic, and supercritical fluid extraction,15 but the two primary methods of extraction from tobacco are countercurrent perfusion-extraction and compression sieving.
Countercurrent perfusion-extraction requires the addition of an externally applied buffer or solvent, which is added to comminuted
solid matter in a continuous process. Proteins are extracted by the liquid solvent phase flowing countercurrent to the solids.
In this type of extraction, there is a continuous gradient of target protein in both the solvent and solids. The solids removed
from the extraction machinery at one end have low target-protein concentrations, while the solvent removed at the opposite
end has a high target-protein concentration. However, the addition of solvent increases the process volume, which requires
larger equipment and adds to raw material costs.
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