Optimizing Co‐Expression of Human Circadian Protein Complex CLOCK/BMAL1

Abstract

Circadian rhythm is the internal clock in the body that cycles in approximately 24 hours. It controls many physiological changes such as the sleep‐wake cycle and the body‐temperature fluctuation. In humans, every single cell has its own circadian rhythm that is synchronized by a master clock in the brain. At the molecular level, circadian rhythms of individual cells are controlled by the transcriptional‐translational feedback loop (TTFL). This loop is initialized by the heterodimerization of circadian locomotor outputs cycle kaput (CLOCK) and brain muscle ARNT‐like 1 (BMAL1). The long‐term goal of the project is to study the structure and the function of the full‐length human CLOCK/BMAL1 complex. Human CLOCK and BMAL1 genes have been cloned into a bacterial expression system. In order to reach the long‐term goal, it is necessary to optimize the condition of expression and purification of the CLOCK/BMAL1 complex to a high quality and homogeneity. In this study, concentration of the inducer, induction time, cell density, and lysis methods have been optimized through multiple series of expression trials. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE) and Western Blotting have been used to assess the quantity and quality of CLOCK/BMAL1 expression. Protein expressed using the optimized conditions has been subjected to purification experiments. Expression and purification of the CLOCK/BMAL1 proteins will build a solid foundation for the structural and functional studies, which will deepen our understanding of the molecular mechanism of circadian rhythm that will facilitate the design of therapeutic reagents to treat circadian related diseases such as sleep‐wake disorder and jet lags.