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Optimization of Protein Extraction from Tobacco

Published by the American Society of Agricultural and Biological Engineers, St. Joseph, Michigan www.asabe.org

Citation:  Paper number  037013,  2003 ASAE Annual Meeting . (doi: 10.13031/2013.15427)
Authors:   Chenming Zhang, David H. Vaughan, Jacob Cartner, J. Mark Jackson, Amber Seba, Rebecca Tanory
Keywords:   Tobacco; Transgenic plants; Protein; Protein extraction

The effects of pH, ionic strength, detergents, chaotropic reagents, and organic solvents on protein extraction were studied to determine optimal conditions for protein extraction from burley tobacco leaves. The bicinchoninic acid (BCA) assay was used to quantify the amount of protein extracted under various conditions, and gel electrophoresis was used to characterize the proteins extracted. In a pH study with pH 3-9 buffers, results indicated a maximum extraction of 1.697 % g protein/g leaf while using a pH 8 buffer. An ionic strength study showed a buffer strength of 0.25 M was optimal for protein extractions at pH 3 and pH 7, yet pH 9 exhibited no evident trend as to a preferable ionic strength. A 15:1 buffer to biomass ratio was found to be optimal for extracting the most soluble protein from tobacco. Outcomes of a detergent study using 0.05% and 0.10% sodium dodecyl sulfate (SDS), Tween-20, and Triton X-100 yielded more protein in solution when compared to extractions made without additives. Approximately 3% g protein/g leaf was obtained using 0.5% and 1.0% SDS, providing the most protein extracted from tobacco during this experiment. Solutions of 0 M, 1 M, and 3 M urea at pH 8 did not vary considerably the amount of protein extracted. Likewise, organic solvents did not increase the amount of protein extracted. Overall optimal extraction conditions for burley tobacco protein occurred at a pH of 8, 0.25 M buffer, and 0.5 % SDS detergent. Gel electrophoresis was used to characterize any differences in proteins extracted under varying conditions.

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