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

Abstract

The circadian rhythm is an internal biological clock composed of different autonomous oscillators that regulate many physiological activities such as the sleep wake cycle. Long term disruption of circadian rhythm can lead to sleep disorders, neurodegenerative disease, metabolic syndrome, and high risks of cancer. The mechanism of circadian rhythm is driven by interlocked time‐delayed feedback loops consisting of positive and negative transcriptional regulators. For instance, Retinoic Acid Receptor‐Related Orphan Receptors (RORs) are nuclear receptors to improve circadian rhythm stability and functionality. A naturally occurring flavonoid Nobiletin (NOB) was reported to enhance circadian amplitude due to its high affinity to human ROR gamma (hRORγ). The interaction between NOB and hRORγ has not been studied in detail which hinders the future application of NOB as a promising candidate of circadian interfering medicine to ameliorate related diseases. The goal of this project is to solve the atomic three‐dimensional structure of hRORγ/Nobiletin complex to understand their interaction. The hRORγ gene was cloned and expressed using E. coli BL21 cells. Expression protocols were optimized to produce large amounts of recombinant hRORγ which have been purified using affinity chromatography and size exclusion chromatography. Circular dichroism has been used to verify that the purified recombinant hRORγ is properly folded. High concentration homogenous recombinant hRORγ will be co‐crystalized with Nobiletin. The atomic structure of the complex will be determined using X‐ray crystallography. The resultant structural information will shed light on Nobiletin’s enhancement mechanism on circadian rhythm and build the foundation for future drug development.