Cytochrome P450 enzymes catalyze key reactions in human drug metabolism and steroid hormone biosynthesis, but they can be difficult to study because these membrane proteins are difficult to generate recombinantly. The human drug-metabolizing P450 CYP3A5 has not been studied in depth due to difficulty with expression in E. coli and purification of this membrane protein. Recently, modification of the amino acid sequence permitted CYP3A5 expression in E. coli, making purification possible. Construction of the CYP3A5-expressing plasmid was accomplished by ligating the modified CYP3A5 cDNA into of the pCWori+ vector. During purification the addition of the tight-binding ligand clotrimazole helped stabilize the CYP3A5 protein, while sonication as a method of cell lysis allowed for a greater recovery of CYP3A5. The human steroidogenic P450 CYP11B1 had recently been expressed as a fusion protein with its redox partner adrenodoxin (Adx) to examine their crucial protein-protein interaction, but protein expression yields were low. Conditions were altered to improve expression levels to permit further assays and crystallization. Promising amounts of Adx/CYP11B1 fusion protein were obtained using autoinduction as a preferred method of induction, and a 24-hour expression period, compared to Terrific Broth media, induction by IPTG, and longer growth periods. In addition, different arabinose concentrations were used to determine the optimal concentration for induction of chaperone proteins that assist with Adx/CYP11B1 folding and improve protein expression. Generation of recombinant protein will permit the study of the structure and functions of important enzymes in human drug metabolism and steroid hormone synthesis.

Recombinant expression of xenobiotic and steroidogenic cytochrome P450 enzymes

2019 Spring National Meeting

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