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Bacterial expression of radio-labeled recombinant proteins for studying AHR signalling

Author

Anthony Benjamin Delucchi, University of the Pacific

Date of Award

2001

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

William K. Chan

First Committee Member

Craig A. Vierra

Second Committee Member

Gregg D. Jongeward

Third Committee Member

Paul A. Richmond

Abstract

The ligand activated transcription factor Aryl Hydrocarbon Receptor (AHR) forms a DNA binding heterodimer with the Aryl Hydrocarbon Nuclear Translocator (ARNT) in response to planar aromatic hydrocarbons. In addition to AHR and ARNT there are at least three other proteins involved in AHR signaling. These proteins are the co-chaperone p23, Ara-9 and two molecules of Heat Shock Protein-90 (HSP-90). This study documents the production of Ara-9 and C∆418 (an ARNT deletion construct) in a modified thioredoxin fusion system. These proteins were expressed in a system that allowed for removal from the fusion partner via a thrombin recognition site as well as the incorporation of an in vitro phosphorylation site. The proteins were then expressed and column purified from E. coli. Once the proteins were expressed and purified they were cleaved from the thioredoxin fusion partner and radioloabeled. Following optimization of the proteolytic digest and radio-labeling each protein was subject to two methods of functional analysis. C∆418 function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with ARNT. In addition the functionality of C∆418 was assessed by co-precipitation showing that the ThioHis-produced C∆418 was indeed able to dimerize with C∆553 (an AHR deletion construct). The ThioHisproduced Ara-9 was also assessed for functionality by EMSA and showed that it was able to restore AHR/ ARNT/DRE complex formation as effectively as Ara-9 produced in a baculovirus system. In addition the function of ThioHis-Ara-9 was also assessed through Far-Western blotting for its ability to associate with renatured HSP-90. These studies involving C∆418 and Ara-9 show that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system In addition this study documents the production of a plasmid (pCMV-Ara-9) for transfection into the HepG2 cell line to monitor the effects of increased cellular Ara-9 on AHR.

Pages

53

Recommended Citation

Delucchi, Anthony Benjamin. (2001). Bacterial expression of radio-labeled recombinant proteins for studying AHR signalling. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/550