Sakaki T, Oeda K, Miyoshi M, Ohkawa H
J Biochem. 1985 Jul;98(1):167-75. doi: 10.1093/oxfordjournals.jbchem.a135255.
Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.
大鼠细胞色素P-450MC cDNA在酵母ADH1启动子和终止子的控制下，在酿酒酵母AH22、SHY3和NA87-11A细胞中表达。尽管用构建的表达质粒pAMC1转化的这三种酵母菌株都含有约三个质粒拷贝，但携带质粒pAMC1的AH22细胞中P-450MC mRNA和相应蛋白质的水平分别比其他两种菌株高1.4至1.7倍和2倍。通过一种快速纯化方法从携带pAMC1的AH22细胞的微粒体部分中纯化了P-450MC蛋白。纯化的P-450MC蛋白的表观分子量、色谱行为、光谱特性、底物特异性和免疫化学特性与大鼠肝脏P-450MC-I和P-450MC-II的这些特性无法区分（佐佐木，T.等人（1984年）《生物化学杂志》96，117 - 126）。纯化蛋白的NH2末端10个残基的氨基酸序列与P-450MC-I和P-450MC-II的相同。此外，对含有pAMC1的AH22细胞微粒体部分的HPLC分析表明，合成的P-450MC蛋白对应于P-450MC-II，而不是P-450MC-I。通过另一种纯化方法，我们获得了缺失完整P-450MC NH2末端30个氨基酸的裂解P-450MC蛋白。裂解的P-450MC对苯并(a)芘和7-乙氧基香豆素的光谱特性和单加氧酶活性与完整P-450MC的几乎相同。
