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Report No.

Purification of cytochrome P450 2C9.1, 2C9.3 and 2C9.30 expressed in ${it E. coli}$ and their activities toward arachidonic acid ${it in vitro}$

Maekawa, Keiko*; Matsuzawa, Yumiko*; Adachi, Motoyasu; Kuroki, Ryota; Saito, Yoshiro*

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the oxidative metabolism of up to 15% of the drugs that undergo phase I metabolism. We previously reported that ${it $^{*}$3}$ and ${it $^{*}$30}$, which were observed at allele frequencies of about 3% and 0.2% in Japanese, respectively. This study was aimed to access their catalytic activities toward endogenous arachidonic acid ${it in vitro}$. The N-terminal modified and C-terminal His-tagged wild-type CYP2C9, CYP2C9$$^{*}$$3 and CYP2C9$$^{*}$$30 were expressed in ${it E. coli}$ Rosetta2 cells. After cells were lysed, proteins were extracted from membranes using Cymal-5 and purified using Ni-affinity and CM-Sepharose ion exchange columns. Both variants exhibited significantly lower Luciferein-H hydroxylation activities than the wild-type. Arachidonic acid were metabolized into bioactive lipids such as 14,15-epoxyeicosatrienoic acid and 19-hydroxyeicosatetraenoic acid, but their production by variant enzymes was much lower than those by the wild-type.



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