Expression and Purification of Human Circadian Protein hRORγ for Structural and Functional Studies

Abstract

The circadian rhythm is an internal biological clock that runs in many living organisms. It regulates the sleep wake cycle among other physiological functions. Long term disruption of this internal clock can cause sleep disorders, metabolic diseases, and high risks of cancer. The circadian rhythm is controlled at molecular level by the transcriptional‐translational feedback loop (TTFL) that involve positive transcriptional activators CLOCK/BMAL1 and negative feedback suppressors PERs/CRYs. The Retinoic Acid Receptor‐Related Orphan Receptors (RORs) function as ligand‐dependent transcription factors which regulate BMAL1 production to improve circadian rhythm stability and robustness. It was shown that the naturally occurring flavonoid Nobiletin can bind to human ROR gamma (hRORγ) and increase its function through a yet unknown mechanism. Understanding the interaction between hRORγ and Nobiletin would provide insights in enhancing the circadian rhythm and ameliorating its related diseases. This project focuses on solving the three‐dimensional structure of hRORγ/Nobiletin complex to establish the mechanism of their interaction. The hRORγ gene was cloned and expressed using E. coli BL21 cells. Expression protocols were optimized to produce large amounts of hRORγ recombinant proteins. The soluble portion of expressed protein was purified using affinity chromatography and size exclusion chromatography, and the purified recombinant hRORγ is properly folded indicating from circular dichroism data. Once the required concentration is reached, the purified hRORγ will be co‐crystalized with Nobiletin. The crystals would be used for structural determination using X‐ray crystallography. The structural information will shed light on Nobiletin’s enhancement mechanism on circadian rhythm and provide guidance in related drug development.Support or Funding InformationResearch reported is supported by: National Research of General Medical Sciences of NIH under award number SC3GM109870. Researcher has used instruments sponsored by the BUILDing SCHOLARS program funded by the National Institute of General Medical Sciences of NIH under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. Researcher is supported by Maximizing Access to Research Careers Grant # T34GM008048