Abstract
The Aryl Hydrocarbon Receptor (AHR) is a ligand-activated transcription factor with various roles, including cell growth and differentiation. AHR was recently found to be involved in various tumours, but its role can be either facilitative or prohibitive depending on the cancer type and the activating ligand. This study focuses on the ligand activation mechanism that determines the function of AHR across different cell types and conditions. Attempts to discern the mechanism through structural analysis of AHR were limited due to the difficulty in expressing and purifying the protein in sufficient concentrations for analysis. Only recently, developments in Cryogenic Electron Microscopy (cryo-EM) have allowed for characterization of the ligand binding domain of AHR, discerning characteristics of ligand interactions. However, it is currently not possible to study AHR ligand binding in detail. In this study, I demonstrate recombinant human and fruit fly (Drosophila melanogaster) AHR expression and purification from Escherichia coli alongside data of interactions with ligands and interacting proteins. High concentrations of monomeric fruit fly AHR have been obtained with subsequent biophysical analysis demonstrating structural characteristics and ligand affinities. Meanwhile, soluble human AHR has been purified in high concentrations albeit in an aggregated form. Experiments involving ligands, interacting proteins, and dialysis provided insights into the aggregate and avenues for generating well-behaved, monomeric protein to explore in the future. Overall, this study provides high concentrations of recombinant, soluble AHR and sets the direction for future optimizations and analyses.