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Cloning the human vitamin D receptor into the pTwin-1 expression vector.
Elaine D Collins1, Aileen Espinoza, Lily Thao-Nhi Le
1Department of Chemistry, San Jose State University, One Washington Square, San Jose, CA 95192-0101, USA. ecollins@science.sjsu.edu
The Journal of Steroid Biochemistry and Molecular Biology
|February 23, 2010
Summary
Researchers developed a novel method to express and purify the human vitamin D receptor (VDR). This technique facilitates the study of VDR binding to vitamin D3 and its analogs, crucial for understanding gene transcription.
Area of Science:
- Molecular Biology
- Endocrinology
- Biochemistry
Background:
- The nuclear vitamin D receptor (VDR) mediates many actions of 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3].
- VDR belongs to a superfamily of nuclear receptors acting as ligand-dependent transcription factors.
- Ligand binding to VDR induces conformational changes, enabling interaction with coactivators to modulate gene transcription.
Purpose of the Study:
- To characterize the binding of VDR to 1,25(OH)2D3 and its analogs.
- To develop an efficient expression and purification system for the human VDR.
Main Methods:
- Cloning the cDNA for human VDR into the pTwin1 expression system.
- Utilizing a chitin-binding peptide and a yeast intein for VDR fusion protein expression.
- Employing a self-cleaving intein for VDR purification from a chitin column via pH change.
Main Results:
- Successfully generated a VDR-fusion protein using the pTwin1 expression system.
- Western blot analysis confirmed the expression of a VDR-fusion protein of approximately 75 kDa.
- The intein-based system allowed for the release of VDR without additional amino acids.
Conclusions:
- The developed expression system provides a method for producing and purifying functional VDR.
- This system is suitable for studying VDR interactions with vitamin D analogs.
- Facilitates further research into VDR-mediated gene regulation.
