Shimada T, Wunsch R M, Hanna I H, Sutter T R, Guengerich F P, Gillam E M
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Arch Biochem Biophys. 1998 Sep 1;357(1):111-20. doi: 10.1006/abbi.1998.0808.
Human cytochrome P450 (P450) 1B1 was expressed in Escherichia coli at a level of 200 nmol/liter culture using a pCW vector by removal of codons 2-4 and modification of the nucleotide sequence of the resulting N-terminal seven codons; a similar level of expression was found with a bicistronic construct that also expressed human NADPH-P450 reductase. P450 1B1 was purified (from the monocistronic system) to electrophoretic homogeneity and a specific content of 9.2 nmol P450/mg protein using DEAE, CM, and hydroxylapatite chromatography. The absolute spectra showed a considerable fraction of high-spin iron and little cytochrome P420. The catalytic activity of the purified enzyme was considerably enhanced in the presence of cholate. Both reconstituted P450 1B1 and the bacterial membranes prepared from the bicistronic vector system had similar7-ethoxyresorufin O-deethylation activities; as expected, 17beta-estradiol was hydroxylated primarily at the 4-position. The ability of human P450 1B1 to activate several heterocyclic amines and polycyclic hydrocarbon dihydrodiols was confirmed with reconstituted P450 1B1 and the P450 1B1 membranes in which NADPH-P450 reductase was coexpressed.
通过去除密码子2 - 4并修饰所得N端七个密码子的核苷酸序列，使用pCW载体在大肠杆菌中表达人细胞色素P450（P450）1B1，表达水平为200 nmol/升培养物；在同时表达人NADPH - P450还原酶的双顺反子构建体中也发现了类似的表达水平。使用DEAE、CM和羟基磷灰石色谱法（从单顺反子系统中）将P450 1B1纯化至电泳纯，比活性为9.2 nmol P450/毫克蛋白质。绝对光谱显示高自旋铁的比例相当大，细胞色素P420很少。在胆酸盐存在下，纯化酶的催化活性显著增强。重组P450 1B1和由双顺反子载体系统制备的细菌膜具有相似的7 - 乙氧基异吩恶唑酮O - 脱乙基活性；正如预期的那样，17β - 雌二醇主要在4位发生羟基化。用重组P450 1B1和共表达NADPH - P450还原酶的P450 1B1膜证实了人P450 1B1激活几种杂环胺和多环烃二醇的能力。
