Date of Graduation

Summer 2021

Degree

Master of Science in Chemistry

Department

Chemistry

Committee Chair

Natasha DeVore

Keywords

orphan cytochrome P450, protein expression, chromatographic purification, binding affinity assays, protein crystallization

Subject Categories

Analytical Chemistry

Abstract

The human cytochrome P450 enzyme 4X1 (CYP4X1) is among the “orphan” cytochrome P450 meaning there is much unknown about its structure, biological function, and regulation in literature study. Scientific work on this protein has primarily been on identifying its physiological functions and expression sites. A recent study found 2 substrates (arachidonic acid and anandamide) catalyzed by the enzyme. Expression in humans appears to be primarily in the brain, prostate, lungs, and neurovascular systems and research has linked the protein as a potential chemotherapy target in brain cancer. So far, only a few inhibitors have been identified. The goal of this research is to characterize CYP4X1. The first step is to express the protein in E. coli JM109 cell using either the pCW or pET28a vector. Protein expression attempts indicated that pET28a vector in JM 109 cells produced a better yield overall. Protein purification was obtained using cobalt and nickel affinity chromatography made possible by a 6-residue histidine tag affixed to the protein’s C-terminal following expression. Protein purification is followed by SDS-PAGE to ascertain purity. Binding affinity assays were obtained for the two previously published substrates, arachidonic acid, and anandamide. Additional compounds that could be ligands based on their structural similarity to arachidonic were also assayed. Purified protein was concentrated and screened with a commercial crystal screen and a crystal hit has been identified with further optimization required.

Copyright

© Olusegun Adeolu Idowu

Open Access

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