Purification of GFP fusion proteins from transgenic plant cell cultures |
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Authors: | Peckham Gabriel D Bugos Robert C Su Wei Wen |
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Institution: | Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Ag. Sci. 218, Honolulu, HI 96822, USA. |
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Abstract: | Green fluorescence protein (GFP) has become a widely used reporter in many areas of life science. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. GFP itself has been purified from recombinant organisms by several methods, often involving unfavorable conditions (e.g., use of organic solvents and/or low pH) that may be destabilizing to some proteins. In this study, we have developed a general recovery scheme that entails a simple three-step purification procedure for GFP fusion proteins produced in tobacco suspension cells, with the intent of maximizing purity and yield under gentle conditions so as to maintain the integrity of the fusion partner. Ammonium sulfate treatment at 30% (v/v) precipitated particulate matter and removed aggregated material while simultaneously maintaining GFP solubility and increasing hydrophobicity. Hydrophobic interaction chromatography was then performed to eliminate the majority of background proteins while eluting GFP and fusions in a low ionic buffer suitable to be directly applied to an ion-exchange column as the final step. Three intracellular proteins, secreted alkaline phosphatase (SEAP), and granulocyte-macrophage colony-stimulating factor (GMCSF), each fused to GFP, as well as GFP itself, were recovered with yields exceeding 70% and purity levels over 80%. This purification scheme exploits the hydrophobic nature of GFP while maintaining a gentle environment for labile fusion partners. Although some optimization may be required, we believe this scheme may serve as a benchmark for purifying other GFP fusion proteins. |
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Keywords: | Chromatography Green fluorescent protein Fusion proteins Tobacco suspension cells Transgenic plants |
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