Abstract 2184 Co-Expression of Human Circadian Protein Complex CLOCK/BMAL1

The circadian rhythm is an internal biological clock that oscillates roughly every 24 hours and is responsible for maintaining many physiological properties such as sleep patterns, body temperature, and eating habits. In humans, the harmonious synchronization of these rhythms across various cell types is orchestrated by a master clock situated in the brain. At the cellular level, the circadian rhythm is governed by a transcription-translation feedback loop (TTFL), initiated by the heterodimerization of the circadian locomotor outputs cycle kaput (CLOCK) and brain muscle ARNT-like 1 (BMAL1) proteins. This study aims to generate a substantial quantity of full-length CLOCK/BMAL1 complexes for comprehensive structural and functional investigations. To achieve this goal, CLOCK and BMAL1 have been cloned into a dual expression vector, allowing for their co-expression. Expression conditions have undergone rigorous optimization, including temperature, inducer concentration, and cell density adjustments. Purification protocol protocol is being fine-tuned to yield highly purified complexes. The successful expression and purification of the CLOCK/BMAL1 complex lay a solid foundation for forthcoming structural determinations and functional analyses. These findings promise to deepen our comprehension of the molecular intricacies underlying circadian rhythms, offering valuable insights into the design of therapeutic agents for circadian-related disorders, such as sleep-wake disorders and jet lag. Research in this poster was supported by the National Research of General Medical Sciences (NIGMS) of NIH under award number SC3GM108870 and funded partially by The Welch Foundation with grant No. AH- 2126-20220331. KAV was supported by the National Research of General Medical Sciences of NIH under the fellowship grant number T34GM145529.