Optimisation of the expression of Sigma Non-Opioid Intracellular Receptor 1 (o1) in membrane mimetic systems

The sigma non-opioid intracellular receptor 1 (σ1) is a highly important, yet poorly understood chaperone membrane protein. Although structural studies have come a long way in identifying the crystal structure and its pharmacological characteristics, the proteins relation to disease and illness is yet to be fully established. Currently a world leading biopharmaceutical company called Anavex, is conducting phase 2b and 3 clinical trials on a compound called A2-73 to target the sigma-1 receptor in Alzheimer's disease. This compound has demonstrated a good safety profile and significant therapeutic effects in some patients by slowing Alzheimer's disease progression and even reversing its development if treated within early stage onset. This research project aimed to develop recombinant protein expression systems for the sigma-1 receptor in C43 Escherichia coli and INVSc Saccharomyces cerevisiae cell lines. In addition, we set out to compare current detergent solubilization techniques with upcoming nanodisc technology, as previous publications have demonstrated undesirable results when conducting structural studies on this protein. Here we report, the successful isolation of the human sigma-1 receptor in C43 expression systems and effective solubilisation using detergent based methods. Unfortunately, we were unable to solubilise this receptor using nanodisc technology in any of the expression system we used during this study. In addition, we were unable to conclude the definitive isolation of the sigma-1 receptor in the INVSc1 cell line due to potential cloning and expression protocol issues. However, we did report positive results for the use of nuclear magnetic resonance (NMR) spectroscopy in ligand interaction experiments such as CPMG studies. From these results we identified a plausible binding interaction between the sigma-1 receptor and the agonist compound fluoxetine hydrochloride. However, this result was not entirely conclusive due to a possible indication of the agonist binding to micelle formation from the use of detergent. Overall, this study highlighted the challenges and potentials of researching the sigma-1 receptor and provides a strong indication for future research.