Saade Evelyne, Mechold Undine, Kulyyassov Arman, Vertut Damien, Lipinski Marc, Ogryzko Vasily
Institut Gustave Roussy, Villejuif, France.
Proteomics. 2009 Nov;9(21):4934-43. doi: 10.1002/pmic.200900206.
We describe a modification of the tandem affinity purification method for purification and analysis of multiprotein complexes, termed here DEF-TAP (for differential elution fractionation after tandem affinity purification). Its essential new feature is the use for last purification step of 6xHis-Ni(++) interaction, which is resistant to a variety of harsh washing conditions, including high ionic strength and the presence of organic solvents. This allows us to use various fractionation schemes before the protease digestion, which is expected to improve the coverage of the analyzed protein mixture and also to provide an additional insight into the structure of the purified macromolecular complex and the nature of protein-protein interactions involved. We illustrate our new approach by analysis of soluble nuclear complexes containing histone H4 purified from HeLa cells. In particular, we observed different fractionation patterns of HAT1 and RbAp46 proteins as compared with RbAp48 protein, all identified as interaction partners of H4 histone. In addition, we report all components of the licensing MCM2-7 complex and the apoptosis-related DAXX protein among the interaction partners of the soluble H4. Finally, we show that HAT1 requires N-terminal tail of H4 for its stable association with this histone.
我们描述了一种对串联亲和纯化方法的改进，用于多蛋白复合物的纯化和分析，在此称为DEF-TAP（串联亲和纯化后的差异洗脱分级分离）。其关键的新特性是在最后一步纯化中使用6xHis-Ni(++)相互作用，这种相互作用能抵抗多种苛刻的洗涤条件，包括高离子强度和有机溶剂的存在。这使我们能够在蛋白酶消化之前使用各种分级分离方案，预计这将提高所分析蛋白质混合物的覆盖率，还能对纯化的大分子复合物的结构以及所涉及的蛋白质-蛋白质相互作用的性质提供额外的见解。我们通过分析从HeLa细胞中纯化的含组蛋白H4的可溶性核复合物来说明我们的新方法。特别地，与RbAp48蛋白相比，我们观察到HAT1和RbAp46蛋白有不同的分级分离模式，它们都被鉴定为H4组蛋白的相互作用伙伴。此外，我们报告了许可MCM2-7复合物的所有组分以及可溶性H4相互作用伙伴中的凋亡相关蛋白DAXX。最后，我们表明HAT1与该组蛋白的稳定结合需要H4的N末端尾巴。
