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Detection of the Ligand Bound State of the Human Vitamin D Receptor: Analyzing the Split Green Fluorescent Protein as a Highly Sensitive Biosensor
Thesis - Campus Access Only
Master of Science (MS)
The human vitamin D receptor (VDR) is a part of the steroid-retinoid-thyroid nuclear receptor superfamily of proteins. The classical mode of action for the VDR is to serve as a nuclear receptor that acts as a transcription factor. The nuclear VDR, following binding of the active form of vitamin D3, 1a,25 dihydroxyvitamin D3 (1a,25(OH)2D3), is known to modulate gene transcription of various genes under the control of vitamin D response elements. Some biological responses due to 1a,25(OH)2D3 occur too quickly for this classical mode of action. As such, a membrane-associated protein must be responsible for the modulation of these rapid response pathways. The VDR has been implicated as capable of acting as both a nuclear receptor and membrane-associated receptor.
In order to determine whether or not the VDR is able to localize at the membrane while bound to a ligand, differentiation between liganded and unliganded receptors would be beneficial. We hypothesized that the use of a split green fluorescent protein-human vitamin D3 receptor fusion protein (sGFP-VDR) would allow for a rapid assay capable of selective imaging of the VDR bound to a ligand. Plasmid constructs were generated using site-directed mutagenesis and basic cloning methods to produce a variety of sGFP-VDR fusion proteins with varying linker sequences between the sGFP fragments and the receptor to determine whether the sGFP could be maintained in the "off" fluorescence state in the absence of ligand. All of the generated sGFP-VDR proteins were successfully expressed in Cos-7 cells, and fluorescence analysis showed all of the fusion proteins exhibited fluorescence in the absence of ligand. Further investigation with sGFP systems that are optimized for reduced self-assembly of the two fragments is required.
Kato, Mallory Mary Hanaye, "Detection of the Ligand Bound State of the Human Vitamin D Receptor: Analyzing the Split Green Fluorescent Protein as a Highly Sensitive Biosensor" (2013). Master's Theses. 4283.