Purification of poly‐ubiquitinated proteins by S5a‐affinity chromatography
Purification of poly‐ubiquitinated proteins by S5a‐affinity chromatography
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DOI：
10.1002/1615-9861(200106)1:6
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发表时间：
2001-06
期刊：
影响因子：
3.4
通讯作者：
R. Layfield;D. Tooth;M. Landon;S. Dawson;J. Mayer;A. Alban
中科院分区：
文献类型：
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作者：
R. Layfield;D. Tooth;M. Landon;S. Dawson;J. Mayer;A. Alban
多聚泛素化是泛素的异肽连接链与其他靶蛋白的翻译后共价缀合，是26 S蛋白酶体复合物进行蛋白水解降解的中心信号。26 S蛋白酶体的S5 a亚基结合含有四个或更多个泛素的多聚泛素链。我们使用固定化谷胱甘肽-S-转移酶（GST）-S5 a融合蛋白从哺乳动物组织中纯化聚泛素化蛋白，旨在扩大泛素途径的已知底物库。在体外泛素化诱导后，从正常猪脑提取物中成功纯化了多泛素化蛋白的复杂混合物。该混合物的二维凝胶的蛋白质印迹显示至少两个对角系列的泛素阳性斑点。每个系列中的单个点相隔约9 kDa，表明它们代表多泛素化蛋白，链中的泛素数量不断增加。从泛素化诱导的人胎盘提取物中纯化的S5 a结合蛋白，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离并通过考马斯染色可视化，包含一个表观变性分子量约为60 kDa的单一主要种类。Edman降解鉴定该蛋白为hHR 23 B，其为酿酒酵母DNA修复蛋白Rad 23 p的人类同源物。在这种情况下，hHR 23 B不是泛素化的，而是在其N末端含有内在的泛素样结构域，通过该结构域，它与S5 a相互作用（Hiyama，H.，例如，1999，274，28 019-28 025）。
Poly‐ubiquitination, the post‐translational covalent conjugation of isopeptide‐linked chains of ubiquitin to other target proteins, is the central signal for proteolytic degradation by the 26S proteasome complex. The S5a subunit of the 26S proteasome binds poly‐ubiquitin chains containing four or more ubiquitins. We have used an immobilised glutathione‐S‐transferase (GST)‐S5a fusion protein to purify poly‐ubiquitinated proteins from mammalian tissues, with the intention of expanding the repertoire of known substrates of the ubiquitin pathway. A complex mixture of poly‐ubiquitinated proteins was successfully purified from normal pig brain extract following induction of in vitro ubiquitination. Western blots of two‐dimensional gels of this mixture showed at least two diagonal series of ubiquitin‐positive spots. Individual spots in each series were separated by approximately 9 kDa suggesting that they represent poly‐ubiquitinated proteins with increasing numbers of ubiquitins in the chains. S5a‐binding proteins purified from ubiquitination‐induced human placental extracts, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualised by Coomassie staining, contained a single major species with an apparent denatured molecular mass of approximately 60 kDa. Edman degradation identified this protein as hHR23B, a human homologue of the Saccharomyces cerevisiae DNA repair protein Rad23p. In this case hHR23B is not ubiquitinated but instead contains an intrinsic ubiquitin‐like domain at its N‐terminus, through which it interacts with S5a (Hiyama, H., et al., J Biol. Chem. 1999, 274, 28 019–28 025).