Protein Studies: The Function of Constitutive Androstane Receptor (CAR) and its Associated Ligands

Constitutive androstane receptor (CAR) is a nuclear receptor implicated in many physiological activities of the cell. Although it has the structural capability to function in the absence of a bound ligand, CAR has a wide variety of known ligands, such as CINPA-1, and the interaction with these ligands is important for possible drug treatment therapies. This study amplified the genetic construct for CAR by polymerase chain reaction (PCR), grafted it into a plasmid, and transformed Escherichia coli strains so that they acquired the plasmid. The strains were then assayed for CAR expression, and the strain with the best expression was grown for large-scale protein expression. The protein was isolated via affinity chromatography and further purified by size-exclusion chromatography. After purification the protein was concentrated and flash-frozen for long-term storage. A crystallization conditions screening was performed to determine the structure of CAR and its interaction with CINPA-1, but this screen was unsuccessful due to equipment malfunction.