Arribas, RaquelAlanezi, Sarah Abdullah2024-12-042024APAhttps://hdl.handle.net/20.500.14154/74014The Per3 gene plays a crucial role in regulating circadian rhythms, profoundly impacting sleep-wake cycles and metabolic processes. Despite its biological significance, the expression and purification of Per3 have presented substantial challenges, often resulting in low yields and poor solubility. This study tackles these obstacles by employing innovative cloning and expression techniques, particularly utilizing a GST-Per3long to enhance solubility and purification efficiency. We designed primers incorporating homology regions complementary to restriction enzyme-digested ends and employed the In-Fusion cloning method to integrate the Per3 gene into the p3E plasmid vector with high precision. The transformation efficiencies were remarkable, with colony counts reaching 2.292 x 10^8 colonies per μg of plasmid DNA. PCR amplification confirmed the successful integration of the Per3 gene, with distinct bands observed at the expected size of 1143 bp, which was further confirmed by DNA sequencing. Protein expression trials identified 25°C as the optimal temperature, significantly improving the yield and solubility of the GST-tagged Per3 protein. Subsequent purification through GST Affinity chromatography and gel filtration chromatography yielded a highly pure protein, as confirmed by SDS-PAGE and native gel electrophoresis. Although the initial yield was modest, the high purity of the purified protein provides a robust foundation for future functional and structural studies. This study not only establishes a reliable protocol for Per3 expression and purification but also opens avenues for investigating the interactions of Per3 with other circadian proteins, such as CRY and BMAL1, to determine their mutual exclusivity and relative affinity. These interactions are crucial for understanding the biological role of Per3 in fine-tuning the circadian clock. Future work should focus on optimizing expression conditions to further increase yield and investigate the intricate biological activity of the Per3 protein within the circadian network.55enCircadian RhythmBiological ClockSleep-Wake CyclePer3 ProteinCircadian Clock ProteinsMolecular CloningRecombinant Protein ExpressionGST-Tag Fusion ProteinEscherichia coliIn-Fusion CloningProtein SolubilityGene ExpressionChronotherapyProtein-Protein InteractionsCellular TimekeepingChronobiologyMolecular BiologyBiochemistryGene RegulationGenetic EngineeringProtein PurificationPCR ValidationCloning TechniquesSoluble Protein ProductionCircadian BiologyCellular RhythmsMolecular TimekeepingBiological RhythmsTranscriptional Feedback LoopsClock Gene InteractionsProtein ComplexesGene-Protein NetworksCircadian DisordersMetabolic RegulationSleep DisordersRhythmic Gene ExpressionChronomedicineCellular OscillatorsClock Protein MechanismsFunctional GenomicsThesis