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Soluble and Functional Overexpression of the Ligand Binding Domain of Mouse Aryl Hydrocarbon Receptor in E.Coli

Author

Himaja Ponnakanti, 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

William Chan

First Committee Member

Miki Park

Second Committee Member

Timothy Smith

Abstract

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor whose toxicity and carcinogenesis is mediated through various polyaromatic hydrocarbons and other environmental pollutants. The role of AhR in carcinogenesis is an area of concern due its altered levels in various tumors. AhR binds structurally diverse ligands and may elicit different responses upon ligand binding. The crystal structure of mouse AhR PAS-A domain was already obtained due to the robustness of mouse AhR in comparison to human. There is a possibility of overexpressing mouse AhR ligand binding domain in its soluble and functional form, which could be used to perform ligand binding studies. This forms the aim of this thesis. Mouse AhR ligand binding domain was constructed as mAhR aa211-384, which was purified under native conditions with the use of 6 histidine tag but soluble overexpression was not possible. Thus a solubility enhancing tag called maltose binding protein (MBP) was used for purification of mAhR aa211-384 under native conditions, which still did not yield soluble overexpression. The strategy was modified to solubilize the protein by denaturation with the use of 8M Urea, which solubilized the protein but included an issue of protein binding to column. Subsequent use of an even stronger denaturant, 6M guanidine hydrochloride, solubilized most of the protein and purified mAhR aa211-384 in huge amount. Successful refolding of mAhR aa211-384 with the help of MBP was made possible by gradual reduction of denaturant in the presence of arginine, but 6 histidine tag failed to refold the protein. The refolded protein was tested for its secondary structure by circular dichroism. Thus, mAhR aa211-384 was solubilized and purified under denaturing conditions with the help of both 6 histidine and MBP, however efficient refolding of mAhR aa211-384 was only possible with the help of MBP but not 6 histidine. The MBP-refolded mAhR aa211-384 stayed in solution even after the removal of 0.1 M arginine, thus confirming the effectiveness of MBP in protein refolding in comparison to 6 histidine tag.

Pages

81

ISBN

9781369797985

Recommended Citation

Ponnakanti, Himaja. (2017). Soluble and Functional Overexpression of the Ligand Binding Domain of Mouse Aryl Hydrocarbon Receptor in E.Coli. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/260

Creative Commons License

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