Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants
Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants
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DOI：
10.1111/j.1365-313x.2004.02031.x
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发表时间：
2004-04-01
期刊：
影响因子：
7.2
通讯作者：
Fromm, ME
中科院分区：
文献类型：
--
作者：
Rohila, JS;Chen, M;Fromm, ME
构建了编码串联亲和纯化（TAP）标签的合成基因，并分析了TAP标签与绿色荧光蛋白（GFP）融合对表达水平和亚细胞定位的影响，以及与杂交糖皮质激素受体融合时对类固醇依赖易位到细胞核和转录的影响。在钙调素结合蛋白（calmodulin-binding protein， CBP）结构域检测到一个核定位信号（nuclear localization signal， NLS），并通过突变去除该信号以提高TAP标签的有效性。此外，还进行了纯化改进，包括抑制共纯化蛋白酶，增加蛋白质交联步骤以提高相互作用蛋白质的回收率。利用改进的合成TAP标记基因和方法分离与杂交糖皮质激素受体相互作用的蛋白，并用质谱法对其进行鉴定。鉴定出的两种蛋白，HSP70和HSP90，已知在哺乳动物细胞体内和体外植物中与糖皮质激素受体相互作用。
A synthetic gene encoding the tandem affinity purification (TAP) tag has been constructed, and the TAP tag assayed for its effects on expression levels and subcellular localization by fusion to green fluorescent protein (GFP) as well as for its effects on steroid-dependent translocation to the nucleus and transcription when fused to a hybrid glucocorticoid receptor. A nuclear localization signal (NLS) was detected in the calmodulin-binding protein (CBP) domain and removed by mutation to improve the usefulness of the TAP tag. Additionally, purification improvements were made, including inhibition of a co-purifying protease, and adding a protein cross-linking step to increase the recovery of interacting proteins. The improved synthetic TAP tag gene and methods were used to isolate proteins interacting with the hybrid glucocorticoid receptor and to identify them by mass spectrometry. The two proteins identified, HSP70 and HSP90, are known to interact with the glucocorticoid receptor in vivo in mammalian cells and in vitro in plants.