Protein Expression, Characterization, and Regulation ofCYP4F4andCYP4F5Cloned from Rat Brain

We previously reported the cDNA cloning of three new forms of P450,CYP4F4, CYP4F5,andCYP4F6,from a rat brain cDNA library. In the present study, we expressedCYP4F4andCYP4F5inEscherichia coliusing the pCWOri expression vector with a modification of their N-terminal amino acid sequences and the incorporation of a C-terminal [His]4tag to aid in purification.CYP4F5recombinant protein was purified to a specific content of 7.7 nmol/mg protein from the membrane fraction ofE. coliand showed ω-hydroxylation activity toward leukotriene B4(LTB4), a chemical mediator of inflammation. On the other hand, the solubilized membrane fraction ofCYP4F4-expressed recombinant protein catalyzed the ω-hydroxylation of prostaglandin A1, prostaglandin E1, and 6-trans-LTB4as well as LTB4. The effects of the peroxisome proliferator, clofibrate, on mRNA expression ofCYP4F4, 4F5,and4F6were studied by Northern blot analysis. The expression levels of the mRNA of theseCYP4Fs were shown to be reduced by clofibrate in liver.