Purification of green fluorescent protein - 1 Purification of Green Fluorescent Protein by Three Phase Partitioning and Hydrophobic Interaction

Purification of green fluorescent protein - 1 Purification...

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Purification of Green Fluorescent Protein by Three Phase Partitioning and Hydrophobic Interaction Chromatography By Estefania Toscano 1
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Abstract Green Florescent Protein has been purified by two different methods in this study. Three- Phase Partitioning (TPP) is the first method and Hydrophobic Interaction Chromatography (HIC) was the second. Purification was followed by a protein characterization analysis using different glucose saturations at different pH levels. Recombinant Green Fluorescent Protein is first expressed by Escherichia coli cells then lysed by sonication following two rounds of the first purification, three phase partitioning. Three phase partitioning uses t-butanol and ammonium sulfate to precipitate enzymes and proteins from aqueous solutions resulting in a 2-fold purification. The second purification, Hydrophobic Interaction Chromatography separates biomolecules, including the protein of interest, based in hydrophobicity. The resin used in the column is Phenyl Sepharose 6 fast flow and the three different buffers, binding, washing, and elution buffers are 2.4M Ammonium Sulfate, 0.3M Sodium Phosphate, and TE buffer. Due to complications, 20% ethanol was used instead of designated buffers to elude GFP from HIC column, half way of the experiment, resulting in a 3.4-fold purification. To finalize the study of Green Fluorescent protein, stability is tested using different pH solutions with different saturations of glucose, concluding this protein is extremely stable and capable of being used for many other research studies under different environments. Introduction Green Fluorescent Protein comes from a jellyfish called Aequorea victoria. Its structure is a beta barrel formed by eleven beta strands with an alpha-helix inside and a chromophore in the middle. This chromophore is responsible for emitting the green fluorescence light. When the protein Aequorin from the jellyfish emits a blue light at 470nm wavelength, the chromophore absorbs it, gets excited, and emits the green fluorescent light at 509nm wavelength. This protein has become very useful for scientific research as a visual marker to study other living cells. Due to the lack of Aequorea victoria jellyfish in the oceans, bacterium E. coli is used as a vector to express GFP through transformation, and run experiments with it. In this experiment, after expressing GFP, it is purified by a two-step process, Three Phase Partitioning and Hydrophobic Interaction Chromatography. Then it is subjected to a stability test. 2
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Three Phase Partitioning (TPP) is a very fast, simple, and effective method used to separate biomolecules and proteins based on their concentration and density. TPP commonly uses t-butanol and ammonium sulfate to precipitate proteins from an aqueous solution. Mixing t- butanol, ammonium sulfate and the sample containing the protein of interest will create three different layers based on density. Depending on the saturation of the ammonium sulfate, the
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