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Strategies of overexpressing retinoid X receptor and pregnane x receptor for functional studies

Author

Chandra Zaneta Bunton, University of the Pacific

Date of Award

2008

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Behavioral Sciences

First Advisor

William K. Chan

First Committee Member

Geoff Lin-Cereghino

Second Committee Member

Joan Lin-Cereghino

Third Committee Member

Lisa A. Wrischnik

Abstract

The ligand activated transcription factor retinoid X receptor (RXR) forms a DNA binding heterodimer with pregnane X rseceptor (PXR) in response to foreign xenobiotics. In addition to RXR and PXR there are other proteins involved in the RXR/PXR signaling pathway. Many proteins involved in this pathway are still unknown. This study documents the production of RXR and PXR in a bacterial recombinant fusion system. These proteins were expressed in a system that allowed purification with six histidine residues.

Once the proteins were expressed and purified from E. coli, they were solublized and tested for function. Different strategies were employed including temperature and inducer studies and denaturing and renaturing techniques to solublize PXR. Following the solubilzation of each protein, all proteins were subjected to a method of functional analysis. RXR function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with PXR. These studies involving RXR and PXR demonstrate that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system.

In addition, this study documents various strategies for combating "inclusion body" formation in the overexpression ofPXR. Also, it describes the production of plasmid pCMV-RXR for transfection into the HepG2 cell line to monitor the levels of cellular RXR in various tissue types.

Pages

63

Recommended Citation

Bunton, Chandra Zaneta. (2008). Strategies of overexpressing retinoid X receptor and pregnane x receptor for functional studies. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/702