Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli
Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli
复制标题
DOI：
10.1016/j.bbrc.2006.03.067
复制
发表时间：
2006-05-26
影响因子：
3.1
通讯作者：
Kagawa, N
中科院分区：
文献类型：
--
作者：
Arase, M;Waterman, MR;Kagawa, N
类固醇 21-羟化酶 P450c21 负责将孕酮和 17 α-羟基孕酮转化为其 21-羟基化衍生物。由于难以获得足够量的纯化蛋白，P450c21 的研究很少。为了解决这个问题，我们尝试在大肠杆菌中以稳定形式表达牛 P450c21。 P450c21 的 N 端膜锚和碱性区域被 CYP2C3 的碱性区域取代。当与分子伴侣 GroES/GroEL 共表达时，工程化的 P450c21 的表达水平高于 1.2 μmol/L 培养物 (> 60 mg/L)。利用三步柱层析，将蛋白质高度纯化至具体含量16.6 nmol/mg（纯度91.2%）。通过凝胶过滤分析确定，纯化的蛋白质在 1% 胆酸钠存在下是单体，这表明 P450c21 的膜锚定截短形式比天然形式更可溶。纯化的酶显示出黄体酮和 17 α-羟基黄体酮的典型底物结合差异光谱和 21-羟化酶活性。膜锚的截短增加了 P450c21 的溶解度，促进该蛋白在大肠杆菌中的表达，从而产生足够数量的生物化学和生物物理研究。 (c) 2006 Elsevier Inc. 保留所有权利。
Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17 alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining Sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Echerichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2 mu mol/L culture (> 60 mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6 nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17 alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E coli yielding sufficient quantities for both biochemical and biophysical studies. (c) 2006 Elsevier Inc. All rights reserved.