Green Fluorescent Protein Purification

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its compact beta-can structure. Biochemists now frequently exploit GFP�s unique structural and physical characteristics to visualize various cellular activities.
One such application of GFP is to understand the role of a hormone, T3, in the translocation of thyroid hormone nuclear receptors (TRs).* In this project, GFP was fused to the human TR subtype b1 protein and the protein�s intracellular movement was visually tracked in live cells in the presence or absence of the T3 hormone. The prevalent hypothesis of this experiment was that T3 is essential in directing TR proteins to the nucleus of the cell where the proteins bind to DNA, regulating thyroid activity.
GFP was fused to the amino terminal because that region is not involved in TRb1�s hormone and transcriptional activity whereas the COOH-terminal region is essential to this protein�s activities. In vitro studies showed that the addition of GFP to TRb1 did not affect TRb1�s ability to bind to T3. Numerous other studies ...

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that the T3 hormone directs the translocation of TRb1 to the nucleus. In the presence of T3, the average nuclear/cytoplasmic distribution ratio of GFP- TRb1 � 5.5 � is much higher than in the absence of T3 � 1.5. The cells transfected with a higher concentration of GFP-TRb1 plasmid showed no difference in nuclear/cytoplasmic ratio. Also, the results from the tests involving the mutants of GFP- TRb1 support the hypothesis that domain D is indeed a nuclear localization signal in TRb1. Approximately 70-80% of the cells containing the GFP-mutant TRb1 showed that the protein remained in the cytoplasm even in the presence of T3. (the other 20-30% of cells showed the GFP-mutant TRb1 to be ...

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