Charles Thomas

Poster Presentation

An Inducible Mammalian Expression System for Studying Recombinant Melanopsin

Intrinsically photosensitive ganglion cells (ipRGCs) are a class of photoreceptors involved in non-image forming functions. The key to these functions is a light-sensitive protein, melanopsin¹. Melanopsin’s structure, reflective of G-protein coupled receptors (GPCRs), provides a technical hurdle in understanding relationships between parts of the molecule. Bioinformatics combined with homologous protein studies suggests a relationship between the proximal C-terminus and intracellular loop 3 (ICL3) with melanopsin’s activated response to light. Electron paramagnetic resonance (EPR) studies are performed to study interactions between these two areas, and the process requires protein samples of recombinant melanopsin upwards of 50uM. An additional technical hurdle is the production of stable mammalian cell lines containing melanopsin. Prior attempts had failed due to the possible premature expression and consequent release of internal calcium stores, preventing the mammalian cells from being maintained. To counter this problem, we present a tightly controlled inducible mammalian expression system. The system, comprised of two plasmids (pERV3/pEGSH) responsible for the induction and expression of recombinant melanopsin, introduced within HEK293 cells is predicted to provide the necessary protein for future EPR studies to elucidate the activation mechanism with respect to melanopsin’s structure.