Optimization of Protein Extraction from Tobacco

Abstract

The effects of pH, ionic strength, detergents, chaotropic reagents, and organicsolvents on protein extraction were studied to determine optimal conditions for protein extractionfrom burley tobacco leaves. The bicinchoninic acid (BCA) assay was used to quantify the amount ofprotein extracted under various conditions, and gel electrophoresis was used to characterize theproteins extracted. In a pH study with pH 3-9 buffers, results indicated a maximum extraction of1.697 % g protein/g leaf while using a pH 8 buffer. An ionic strength study showed a buffer strengthof 0.25 M was optimal for protein extractions at pH 3 and pH 7, yet pH 9 exhibited no evident trendas to a preferable ionic strength. A 15:1 buffer to biomass ratio was found to be optimal forextracting the most soluble protein from tobacco. Outcomes of a detergent study using 0.05% and0.10% sodium dodecyl sulfate (SDS), Tween-20, and Triton X-100 yielded more protein in solutionwhen compared to extractions made without additives. Approximately 3% g protein/g leaf wasobtained using 0.5% and 1.0% SDS, providing the most protein extracted from tobacco during thisexperiment. Solutions of 0 M, 1 M, and 3 M urea at pH 8 did not vary considerably the amount ofprotein 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 proteinsextracted under varying conditions.