Expression and Purification of Cytochrome 2U1 and Canine 1A2 and Metabolism of Caffeine by Cytochrome 1A2
Date of Graduation
Master of Science in Chemistry
Chemistry and Biochemistry
Cytochrome P450 enzymes are a large family of membrane heme proteins involved in eliminating drugs and the synthesis or elimination of steroids, eicosanoids, and vitamins. CYP2U1 is one of the poorly characterized P450 enzymes classified as orphan cytochrome P450 with unknown exact biological function. It is expressed in the brain and thymus as a catalyst in the hydroxylation of arachidonic acid. Unlike other cytochrome P450s, CYP2U1 is a bit unusual with forty extra amino acids. If a mutation occurs, it can lead to an early onset inherited disorder of the central nervous system called Hereditary Spastic Paraplegias (HSP). Most of the P450 enzymes in humans are known and characterized based on their structure and functions. Many of them have orthologous proteins in other species. CYP1A2 metabolizes many substrates like phenacetin, caffeine, acetaminophen, and theobromine (from chocolate) which makes it likely that there are differences between the activity of the canine and human CYP1A2 enzyme. The human CYP1A2 protein is well characterized and has a crystal structure available. This has implications in the treatment of canines in veterinary medicine and in drug metabolism studies, which are sometimes conducted in dogs. Few studies have been conducted to look at the biochemistry of the purified canine CYP1A2. Synthesis of the canine CYP1A2 gene and human CYP 2U1 followed by cloning into the pCW plasmid was done and grown in E. coli JM109 cells for the production of large amounts of proteins that were isolated by breaking the cells open through sonication, solubilized with the detergent CHAPS, and purified with Nickel affinity and ion-exchange chromatography. The purity of the protein after purification was measured by gel electrophoresis and UV-vis chromatography. The purified CYP 1A2 protein was used to characterize the metabolism of caffeine, a substance known to be metabolized by human CYP1A2.
cytochrome p450 enzymes, cyp 1a2, cyp 2u1, arachidinic acid, mutation, hereditary spastic paraplegias
© Brenda M. Wekesa
Wekesa, Brenda M., "Expression and Purification of Cytochrome 2U1 and Canine 1A2 and Metabolism of Caffeine by Cytochrome 1A2" (2021). MSU Graduate Theses. 3706.