Expression and purification of a recombinant form of human aromatase from Escherichia coli
Expression and purification of a recombinant form of human aromatase from Escherichia coli
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DOI：
10.1016/s0006-2952(02)01361-8
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发表时间：
2002-11-01
影响因子：
5.8
通讯作者：
Chen, S
中科院分区：
文献类型：
--
作者：
Zhang, FM;Zhou, DJ;Chen, S
芳香酶将雄激素转化为雌激素，雌激素是一种在乳腺癌的发展中发挥重要作用的激素。芳香酶抑制剂已被证明是治疗雌激素依赖性乳腺癌的有效内分泌疗法。芳香酶的结构功能研究可以为设计高效且特异性的抑制剂提供关键的结构信息。然而，芳香酶结构功能研究因缺乏纯化蛋白质而受到阻碍。在本报告中，我们描述了使用pET载体系统在大肠杆菌中构建和表达人芳香酶重组衍生物，以及通过镍-琼脂糖亲和层析纯化该酶。我们检查了芳香酶全长 Del-38、C-6xHis 标记的 Del-38 和 NC-6xHis 标记的 Del-38 形式的表达。发现氨基末端没有前 38 个氨基酸（即 Del-38）的重组芳香酶比全长酶具有更高的活性。此外，在氨基和羧基末端添加两个单独的六聚组氨酸标签（即 NC-6xHis 标签的 Del-38）增加了重组酶与镍琼脂糖的结合亲和力。用 80 mM EDTA 从镍琼脂糖中洗脱表达的芳香酶（即 NC-6xHis 标记的 Del-38 芳香酶）。 80 mM EDTA 洗脱级分的总芳香酶活性显着高于去污剂溶解的蛋白质提取物，表明镍-琼脂糖亲和层析过程中存在复性过程。当通过 SDS-PAGE 分析时，纯化的芳香酶显示出单条带，并且使用氚水释放测定获得了高达 5.8 nmol/mg/min 的活性。通过酶动力学分析确定雄烯二酮的 K-m 值为 62 +/- 24 nM。还通过减少CO差谱分析、反应产物提取测定和使用两种芳香酶抑制剂（来曲唑和阿那曲唑）的抑制研究来表征重组芳香酶制剂。结果表明，来自大肠杆菌的重组芳香酶具有与人体组织中表达的酶相同的催化特性，对于芳香酶的进一步结构功能研究将非常有用。 (C) 2002 Elsevier Science Inc. 保留所有权利。
Aromatase converts androgen to estrogen, a hormone that plays an important role in the development of breast cancer. Aromatase inhibitors have been shown to be a useful endocrine regimen for estrogen-dependent breast cancer. Structure-function studies of aromatase can generate critical structural information for designing highly potent and specific inhibitors. However, aromatase structure-function studies have been hampered by a lack of purified protein. In this report, we describe the construction and expression of a recombinant derivative of human aromatase in Escherichia coli using the pET vector system, and the purification of the enzyme by means of nickel-agarose affinity chromatography. We examined the expression of the full-length, Del-38, C-6xHis-tagged Del-38, and NC-6xHis-tagged Del-38 forms of aromatase. The recombinant aromatase without the first 38 amino acids from the amino-terminus (i.e. Del-38) was found to have a higher activity than the full-length enzyme. Moreover, the addition of two separate hexameric histidine tags at both the amino and the carboxyl-termini (i.e. NC-6xHis-tagged Del-38) increased the binding affinity of the recombinant enzyme to the nickel-agarose. The expressed aromatase (i.e. NC-6xHis-tagged Del-38 aromatase) was eluted from the nickel-agarose with 80 mM EDTA. The total aromatase activity of the 80 mM EDTA-eluted fractions was significantly higher than the detergent-solubilized protein extract, indicating a renaturation process during the nickel-agarose affinity chromatography. Purified aromatase exhibited a single band when analyzed by SDS-PAGE, and activity up to 5.8 nmol/mg/min was obtained using the tritiated water release assay. The K-m value for androstenedione was determined to be 62 +/- 24 nM by enzyme kinetic analysis. The recombinant aromatase preparation was also characterized by reduced CO-difference spectral analysis, reaction product extraction assay, and inhibition studies using two aromatase inhibitors (letrozole and anastrozole). The results indicate that the recombinant aromatase from E. coli has catalytic properties identical to those of the enzyme expressed in human tissue and will be very useful for further structure-function studies of aromatase. (C) 2002 Elsevier Science Inc. All rights reserved.