Abstract 2090 Bacterial expression and chromatography purification of a human period 2 domain

The circadian rhythm, also known as the biological clock, is an endogenous system that oscillates within an approximately 24-hour cycle. The rhythm synchronizes the physiological, hormonal, and behavioral aspects of the body. Long term disruption of the rhythm may lead to sleep disorders, cognitive underperformance, and high risks of cancers. The rhythm is regulated at the cellular level by a transcriptional translational feedback loop (TTFL), which is constituted by four core proteins: Brain Muscle ARNT Like-1 (BMAL-1), Circadian Locomotor Output Cycles Kaput (CLOCK), Cryptochrome (CRY), and Period (PER). In the human body, it is known that there are three paralogs of PER, in which PER2 is the most relevant for the TTFL. This project focuses on one of the Per-Ant-Sim (PAS) domains of the human Period 2 (hPER2t), which is known to be a molecular sensor for the hPER2. The purpose of this investigation is to obtain the molecular structure of the hPER2 domain (hPER2t). To achieve this, the hPER2t coding sequence had been codon optimized, synthesized, cloned, and transformed into a bacterial expression vector. Optimal conditions were established to produce high quantities of hPER2t. These conditions include cell density, culture temperature, and the inducer concentration. The harvesting protocol has also been optimized in buffer receipts and lysing reagents. The harvested samples will then be purified utilizing various chromatographic techniques prior to utilizing structural studies. Understanding the molecular structure of a hPER2t paves the way for investigating its function and mechanism of molecular interactions with other circadian proteins. This poster presents research supported by NIH grants RL5GM118969, TL4GM118971, UL1GM118970, and SC3GM109870. Additional funding from The Welch Foundation (AH-2126-20220331). Roberto Garza is being funded by the COURI MERITUS program funded by the Vice President of Student Affairs and the Campus Office of Undergraduate Research Initiative (COURI) at UTEP.