Abstract:
Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17β-hydroxysteroid dehydrogenase type 1 isoform (17βHSD1) catalyzes NADPH-dependent conversion of estrone to estradiol, a more potent estrogen, and represents potential drug target for breast cancer treatment. To provide active enzyme for inhibitor screening, 17βHSD1 is usually expressed in insect or mammalian cells, or isolated from human placenta. In the present study we describe a simple protocol for expression and purification of active human 17βHSD1 from BL21(DE3) Escherichia coli cells. Soluble human 17βHSD1 was expressed using a pET28a(+)-based plasmid, which encodes a hexahistidine tag fused to the N-terminus of the protein, and purified by nickel affinity chromatography. The enzyme activity of purified 17βHSD1 was verified by three methods: thin-layer chromatography, an alkali assay and a spectroscopic assay. These non-radioactive enzyme assays require only standard laboratory equipment, and can be used for screening compounds that modulate 17βHSD1 activity.
摘要:
乳腺癌细胞的生长通常依赖于类固醇激素的存在。17β-羟基类固醇脱氢酶1型同工型(17βHSD1)催化NADPH依赖性的雌酮转化为雌二醇,更有效的雌激素,并代表了乳腺癌治疗的潜在药物靶标。为抑制剂筛选提供活性酶,17βHSD1通常在昆虫或哺乳动物细胞中表达,或从人胎盘中分离出来。在本研究中,我们描述了从BL21(DE3)大肠杆菌细胞中表达和纯化活性人17βHSD1的简单方案。使用基于pET28a(+)的质粒表达可溶性人17βHSD1,编码与蛋白质N末端融合的六组氨酸标签,并通过镍亲和层析纯化。纯化的17βHSD1的酶活性通过三种方法进行验证:薄层色谱法,碱测定和光谱测定。这些非放射性酶检测只需要标准的实验室设备,并且可用于筛选调节17βHSD1活性的化合物。