Young C Y, Qiu S D, Prescott J L, Tindall D J
Department of Urology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Mol Endocrinol. 1990 Dec;4(12):1841-9. doi: 10.1210/mend-4-12-1841.
The recent cloning of human androgen receptor (AR) cDNAs in this and other laboratories has provided valuable probes for investigating the structure and function of the AR at the molecular level. We now report the overexpression of a region of the human AR containing both the DNA- and hormone-binding domains in E. coli, which provides a means to produce large amounts of AR for analysis and use in functional studies. Under isopropyl-beta-D-thiogalactopyranoside induction, a tripartite protein, consisting of beta-galactosidase, a collagenase recognition site, and AR polypeptide, was produced in E. coli JM109 using pSS20 a as a vector. About 1 mg of the fused AR could be recovered per liter bacterial culture. The induced protein could readily be detected in a sodium dodecyl sulfate-polyacrylamide gel by Coomassie blue staining. Its identity was confirmed by Western blot analysis using antibodies to both beta-galactosidase and the AR. Scatchard analysis of the androgen-binding activity of the hybrid AR revealed high affinity binding to the synthetic androgen, Mibolerone (Kd, approximately 1.2 nM). Competition studies demonstrated the fusion protein's specificity for androgens. The hybrid receptor formed immune complexes with human anti-AR serum that sedimented at about 19S in 10-50% linear sucrose gradients containing 0.4 M KCl. Gel band shift assays revealed that the hybrid receptor protein forms specific complexes with a synthetic steroid response element derived from the mouse mammary tumor virus long terminal repeat region. These results demonstrate that the recombinant AR expressed in E. coli possesses many of the functional properties characteristic of DNA- and steroid-binding domains of the native AR.
近期，本实验室及其他实验室成功克隆出人类雄激素受体（AR）的cDNA，这为在分子水平研究AR的结构与功能提供了宝贵的探针。我们现在报告在大肠杆菌中过表达包含DNA结合域和激素结合域的人类AR区域，这为大量生产用于分析和功能研究的AR提供了一种方法。在异丙基-β-D-硫代半乳糖苷诱导下，使用pSS20a作为载体，在大肠杆菌JM109中产生了一种由β-半乳糖苷酶、胶原酶识别位点和AR多肽组成的三联体蛋白。每升细菌培养物可回收约1毫克融合的AR。通过考马斯亮蓝染色，可在十二烷基硫酸钠-聚丙烯酰胺凝胶中轻松检测到诱导蛋白。使用针对β-半乳糖苷酶和AR的抗体进行蛋白质印迹分析，证实了其身份。对杂交AR的雄激素结合活性进行Scatchard分析，结果显示其对合成雄激素米勃龙具有高亲和力结合（解离常数Kd约为1.2 nM）。竞争研究证明了融合蛋白对雄激素的特异性。在含有0.4 M KCl的10 - 50%线性蔗糖梯度中，杂交受体与人类抗AR血清形成了沉降系数约为19S的免疫复合物。凝胶迁移率变动分析表明，杂交受体蛋白与源自小鼠乳腺肿瘤病毒长末端重复区域的合成类固醇反应元件形成了特异性复合物。这些结果表明，在大肠杆菌中表达的重组AR具有天然AR的DNA结合域和类固醇结合域的许多功能特性。
