EXPRESSION OF MOUSE FULL-LENGTH ARYL HYDROCARBON RECEPTOR AND HUMAN ARYL HYDROCARBON RECEPTOR LIGAND BINDING DOMAIN IN PICHIA PASTORIS

Author

Yiyuan Wang, University of the PacificFollow

Date of Award

2022

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

William K. Chan

First Committee Member

Miki

Second Committee Member

Geoff Lin-Cereghino

Abstract

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates biological responses to planar aromatic hydrocarbon. AHR activates gene transcription by binding to its corresponding enhancer with its partner-aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, this receptor has been shown to regulate xenobiotic-metabolizing enzymes such as cytochrome P450. AHR exists widely in body tissues and affects bioactivation of carcinogenic compounds, T cell differentiation, fatty acid synthesis, cell proliferation, hematopoietic stem cell differentiation, respiratory reactivity, and insulin sensitivity. Although the precise mechanism illustrating the endogenous AHR function remains unclear, there has been intense interest in exploring AHR as a potential target for the treatment of diseases such as cancer and autoimmune diseases. It is known that mouse ahr d-allele possesses low ligand-binding affinity, whereas mouse ahr b-allele has a higher ligand-binding affinity. The d-allele functions more similarly to human AHR than the b-allele, which is most commonly studied. Human AHR can be rather difficult to study since it is relatively unstable and less sensitive to some ligands in vitro. Thus we generated a deletion construct which has the ligand-binding domain of human AHR and hoped that the expression yield could be increased.

Here, I present the process and the results of expressing the mouse full-length b-allele of AHR and the human AHR ligand binding domain (LBD, amino acids 108 to 400) in Pichia pastoris. A higher enrichment of the b-allele and LBD was observed in wild-type yeast (yJC100) strain when compared to the protease-deficient yeast (ySMD1163) strain. This observation was consistent with the increased copy number in the wild-type strain. Although the LBD transcript was detected in both the wild-type and protease-deficient strains, the LBD protein was only detected in the wild-type strain.

Pages

65

Recommended Citation

Wang, Yiyuan. (2022). EXPRESSION OF MOUSE FULL-LENGTH ARYL HYDROCARBON RECEPTOR AND HUMAN ARYL HYDROCARBON RECEPTOR LIGAND BINDING DOMAIN IN PICHIA PASTORIS. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/3801