Kungulovski Goran, Kycia Ina, Tamas Raluca, Jurkowska Renata Z, Kudithipudi Srikanth, Henry Chisato, Reinhardt Richard, Labhart Paul, Jeltsch Albert
Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany;
Active Motif, Carlsbad, California 92008, USA;
Genome Res. 2014 Nov;24(11):1842-53. doi: 10.1101/gr.170985.113. Epub 2014 Oct 9.
Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
组蛋白的翻译后修饰（PTM）构成了一种主要的染色质索引机制，对其进行恰当的表征具有至关重要的生物学意义。到目前为止，尽管存在诸如批次间特异性和结合亲和力的变异性等问题，PTM特异性抗体一直是研究组蛋白PTM的标准试剂。在此，我们成功地利用天然存在的和工程化的组蛋白修饰相互作用结构域来检测和鉴定组蛋白PTM以及不同类型染色质的类似染色质免疫沉淀（ChIP）的富集。我们的结果表明，组蛋白相互作用结构域是强大且高度特异性的试剂，能够替代或补充组蛋白修饰抗体。这些结构域可以在大肠杆菌中以低成本和恒定质量进行重组生产。阅读结构域的蛋白质设计允许产生新的特异性、添加亲和标签以及制备作为匹配阴性对照的PTM结合口袋变体，而这对于抗体来说是不可能的。
