A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta
A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta
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特异性RNA结合蛋白的高产率亲和纯化方法：从人胎盘中分离铁调节因子
DOI：
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发表时间：
1990
期刊：
影响因子：
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通讯作者：
L. Kühn
中科院分区：
文献类型：
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作者：
B. Neupert;N. Thompson;C. Meyer;L. Kühn
我们描述了一种亲和纯化特定 RNA 结合蛋白的简单方法。与 RNA 的蛋白质结合位点相对应的 DNA 序列被亚克隆到 T7 病毒启动子和 Poly(A) 轨道之间的体外转录载体中。聚腺苷酸化 RNA 转录物与聚 (U)-琼脂糖结合，随后与肝素-琼脂糖上预纯化的细胞提取物一起孵育。通过高盐洗脱从亲和基质中以高产率和高纯度回收特异性吸附的蛋白质。使用这种方法，我们分离了铁调节因子 (IRF)，这是一种细胞质蛋白，分别与铁蛋白和转铁蛋白受体 mRNA 5' 和 3' 非翻译序列中的特定回文元件结合。该调节因子的激活和结合与转铁蛋白受体 mRNA 稳定性的增加和铁蛋白翻译的抑制相关。从人胎盘中纯化的因子在凝胶色谱中以单体形式迁移，但通过 SDS/PAGE 分析时，以等摩尔量的两种分子量为 95 和 100 kDa 的蛋白质存在。根据其等电点的一致性及其形成 RNA-蛋白质复合物的特异性来判断，这两种蛋白质高度相关。
We describe a simple method for the affinity purification of specific RNA-binding proteins. DNA sequences corresponding to the protein-binding site of the RNA are subcloned into an in vitro transcription vector between the T7 viral promoter and a poly(A) track. A polyadenylated RNA transcript is bound to poly(U)-Sepharose and subsequently incubated with a cellular extract prepurified on heparin-agarose. Specifically adsorbed proteins are recovered in high yield and purity from the affinity matrix by high salt elution. Using this method we isolated the iron regulatory factor (IRF), a cytoplasmic protein which binds to specific palindromic elements in the 5' and 3' untranslated sequences of ferritin and transferrin receptor mRNA, respectively. Activation and binding of this regulatory factor correlates with increased transferrin receptor mRNA stability and inhibition of ferritin translation. The purified factor from human placenta migrates as a monomer in gel chromatography, but is present in equimolar amounts of two proteins with molecular weights of 95 and 100 kDa when analysed by SDS/PAGE. The two proteins are highly related as judged by the identity of their isoelectric points and their specificity to form RNA-protein complexes.