Express and Purify Human CRY2 for Functional Analyses

Abstract

The circadian rhythm is a biological clock responsible for regulating the daily cycles of essential physiological and metabolic processes, such as sleep wake cycles, by responding to light and dark changes. Every cell has its own circadian rhythm that is synchronized by a master clock in the brain. At the cellular level, circadian rhythms are controlled by a time‐delayed transcriptional‐translational feed‐back loop (TTFL), which consists of several core proteins. These proteins include circadian locomotor output cycles protein kaput (CLOCK), brain and muscle ARNT‐like 1 (BMAL1), cryptochrome (CRY), and period (PER). CLOCK and BMAL1 initiate the TTFL by activating the transcription of several circadian genes including CRYs and PERs that will be translated in the cytosol. With accumulation after a certain time, CRYs/PERs complexes will translocate back to the nucleus to negatively inhibit CLOCK/BMAL1 activities to finish the TTFL. There are two paralogs of CRY within the human genome: CRY1 and CRY2, both of which have essential roles in the negative feedback. The purpose of this project is to study the structure and function of human CRY2. Regardless of many advances in this field including available structure of CRYs, the detailed interaction between CRY2 to other circadian core proteins are still limited. Through this research we would be able to express and purify large quantities of hCRY2 that will be used for the future interaction studies. The results will deepen our understanding of the mechanisms behind circadian rhythm and its associated diseases, facilitating the development of prevention and treatment strategies.