Rapid purification of protein complexes from mammalian cells.
Rapid purification of protein complexes from mammalian cells.
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从哺乳动物细胞中快速纯化蛋白质复合物。
DOI：
10.1093/nar/28.12.e61
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发表时间：
2000
影响因子：
14.9
通讯作者：
Germino,J
中科院分区：
文献类型：
--
作者：
Medina,D;Moskowitz,N;Khan,S;Christopher,S;Germino,J
对生物学上重要的蛋白质的蛋白质结合配偶体的评估是目前热门研究的领域，特别是自从酵母双杂交测定的发展以来。然而，并非该测定发现的所有蛋白质-蛋白质相互作用都具有生物学相关性，必须进行另一项验证测定。理想情况下，这种测定应该是快速的、通用的，并且在尽可能模仿“正常”生理状态的条件下进行。为了实现这一目标，我们构建了两种真核表达载体，有助于从哺乳动物细胞中纯化目的蛋白以及任何相关蛋白。这些载体具有以下特征：（i）四环素响应启动子，以便可以调节蛋白质生产的水平； (ii) N-末端谷胱甘肽S-转移酶标签或HA1表位的三重重复，以促进分别通过谷胱甘肽亲和层析或免疫沉淀纯化蛋白质，然后是多克隆位点； (iii) 增强型绿色荧光蛋白的基因（用于检测融合蛋白的存在和亚细胞定位）； (iv)用于选择稳定转化体的嘌呤霉素标记； (v)用于载体游离复制的截短的EBNA蛋白和oriP序列。后两个特征允许在嘌呤霉素选择下扩增转染细胞的小培养物，从而增加可纯化的标记蛋白的量。我们证明这些载体可用于指导标记蛋白的多西环素诱导表达，并从 HeLa 细胞中回收标记的 CIP1-p21 蛋白复合物。此外，我们发现这些标记的 p21 纯化复合物含有细胞周期蛋白 A 和 Cdk2，已知它们与 p21 相互作用，但不与 β-肌动蛋白相互作用。
The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathioneS-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin.