Kawashima H, Kusunose E, Thompson C M, Strobel H W
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77225, USA.
Arch Biochem Biophys. 1997 Nov 1;347(1):148-54. doi: 10.1006/abbi.1997.0342.
We previously reported the cDNA cloning of three new forms of P450, CYP4F4, CYP4F5, and CYP4F6, from a rat brain cDNA library. In the present study, we expressed CYP4F4 and CYP4F5 in Escherichia coli using the pCWOri expression vector with a modification of their N-terminal amino acid sequences and the incorporation of a C-terminal [His]4 tag to aid in purification. CYP4F5 recombinant protein was purified to a specific content of 7.7 nmol/mg protein from the membrane fraction of E. coli and showed omega-hydroxylation activity toward leukotriene B4 (LTB4), a chemical mediator of inflammation. On the other hand, the solubilized membrane fraction of CYP4F4-expressed recombinant protein catalyzed the omega-hydroxylation of prostaglandin A1, prostaglandin E1, and 6-trans-LTB4 as well as LTB4. The effects of the peroxisome proliferator, clofibrate, on mRNA expression of CYP4F4, 4F5, and 4F6 were studied by Northern blot analysis. The expression levels of the mRNA of these CYP4Fs were shown to be reduced by clofibrate in liver.
我们先前报道了从大鼠脑cDNA文库中克隆出三种新形式的细胞色素P450，即CYP4F4、CYP4F5和CYP4F6的cDNA。在本研究中，我们使用pCWOri表达载体在大肠杆菌中表达CYP4F4和CYP4F5，对它们的N端氨基酸序列进行了修饰，并在C端引入了[His]4标签以辅助纯化。CYP4F5重组蛋白从大肠杆菌的膜组分中纯化至特定含量为7.7 nmol/mg蛋白，并对白三烯B4（LTB4，一种炎症化学介质）表现出ω-羟化活性。另一方面，表达CYP4F4的重组蛋白的溶解膜组分催化前列腺素A1、前列腺素E1、6-反式-LTB4以及LTB4的ω-羟化。通过Northern印迹分析研究了过氧化物酶体增殖剂氯贝丁酯对CYP4F4、4F5和4F6 mRNA表达的影响。结果显示，氯贝丁酯可降低肝脏中这些CYP4F的mRNA表达水平。
