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Overexpression of Human Aryl Hydrocarbon Receptor in E.coli Using Two Different Solubility Enhancing Tags

Author

Sravanthi Tejomurthula, University of the Pacific

Date of Award

2017

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Willian Chan

First Committee Member

Miki Park

Second Committee Member

Timothy Smith

Abstract

Dioxins such as TCDD are environment pollutants whose toxic effects are mediated via aryl hydrocarbon receptor (AhR) signaling pathway. AhR is a ligand sensitive transcription factor. The unbound AhR resides in cytoplasm as a complex containing p23, Hsp90 and XAP2. Upon ligand binding, AhR undergoes conformational change and translocates into the nucleus. Once the AhR dimerizes with AhR nuclear translocator (Arnt), the chaperone proteins in the complex get dissociated followed by the activation of transcription of various genes such as CYP1A1 and CYP1A2 by AhR-ARNT heterodimer. Various cancers have altered levels of AhR in the absence of ligand. Our current knowledge is only limited in the regulation of AhR protein levels in its ligand bound state. However, the mechanism involved in the regulation of AhR protein levels in the absence of ligand is still unknown. To make the study of AhR signaling pathway possible, our lab has been working on the expression of various AhR constructs in E.coli using recombinant DNA technology. As AhR forms inclusion bodies due to its poor solubility in the cytoplasm of the host bacteria, it is tagged as a “difficult to express” protein. Therefore, it is challenging to generate functional recombinant AhR protein. My thesis documents the expression of human AhR construct amino acid 108-400 using two different solubility enhancing tags (thioredoxin and maltose binding protein). Western blot data revealed that the soluble expression of the human AhR construct by thioredoxin solubility enhancing tag has outperformed the other.

Pages

53

ISBN

9781369790795

Recommended Citation

Tejomurthula, Sravanthi. (2017). Overexpression of Human Aryl Hydrocarbon Receptor in E.coli Using Two Different Solubility Enhancing Tags. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/261

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