Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography.
Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography.
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通过序列特异性 DNA 亲和层析快速纯化雌激素受体。
DOI：
10.1021/bi00448a001
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Bambara,RA
中科院分区：
文献类型：
--
作者：
PealeJr,FV;Ishibe,Y;Klinge,CM;Zain,S;Hilf,R;Bambara,RA
摘要：利用序列特异性DNA亲和树脂快速纯化了小牛子宫雌激素受体（ER），使其接近均匀性。通过使用含有8个一致雌激素反应元件（ERE） DNA序列的DNA- sepharo4，实现了对雌激素受体的非常高的选择性。该方案制备的高纯度内质网可以用高比活性的配体经济地标记。该纯化方案选择了在雌激素结合和dna结合域中保留功能的完整受体。纯化后的受体具有分子量为68000的电泳迁移率，在蔗糖梯度上以5S形态沉积，并与人雌激素受体特异性抗体发生反应。^^分离雌激素受体蛋白（ER）的分离方案通常依赖于雌激素亲和层析提供的非常高的选择性。然而，含有高浓度高亲和力雌激素的树脂也使纯化的内质网的后续洗脱变得困难。这个问题的各种解决方案已经被描述。从雌二醇亲和树脂（Sica & Bresciani, 1979; Greene et al., 1980）中提取内质网需要硫氰酸盐（Molinari et al., 1977; Kumar et al., 1978）等异向剂与雌二醇的微摩尔浓度相结合。然而，这种处理也促进内质网二聚体解离成单体亚基（Sica et al., 1976; Weichman & Notides, 1979），需要进一步的步骤来确定纯化后的内质网保留dna结合能力等特性的比例。另外，含有低亲和力雌激素配体的树脂，如雌酮，可以洗脱具有高亲和力17/3-雌二醇的内质酶，而无需使用共向剂（Ratajczak & hannel, 1980; Atrache et al., 1985）。对于这两种方法，使用高ra-
Revised Manuscript Received August 25, 1989 abstract: Rapid purification ofcalf uterine estrogen receptor (ER) to near homogeneity has been ac-complished by use of sequence-specific DNA affinity resin. Very high selectivity for theestrogen receptor is achieved through the use of DNA-Sepharosecontaining eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-bindingand DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68 000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.^^ irification schemes for isolating the estrogen receptor protein (ER) 1have generally depended on the very high se-lectivity offered by estrogen affinity chromatography. How-ever, resins containing high concentrations of high-affinity estrogens also make difficult the subsequent elution of the purified ER. Varioussolutions to this problem have been described. Elution of ER from estradiol affinity resins (Sica & Bresciani, 1979; Greene et al., 1980) requires a chaotropic agent such as thiocyanate (Molinari et al., 1977; Kumar et al., 1978), in combinationwith micromolar concentrations of estradiol. However, such treatment also promotes dissociation of the ER dimer into monomeric subunits (Sica et al., 1976; Weichman & Notides, 1979), necessitating further steps to determine the fraction of purified ERretaining such properties as DNA-binding ability. Alternatively, resins containing low-affinity estrogenic ligands, such as estrone, allow elution of ER with higher affinity 17/3-estradiol, without the use of chaotropic agents (Ratajczak & Hahnel, 1980; Atrache et al., 1985). With both of these approaches, the use of high ra-
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DOI：
--
发表时间：
1983
期刊：
Nippon Ganka Gakkai zasshi
影响因子：
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作者：
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DOI：
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发表时间：
1954
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影响因子：
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作者：
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Payne Bf
影响因子：
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作者：
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DOI：
--
发表时间：
1963
期刊：
Transactions of the American Ophthalmological Society
影响因子：
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