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Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes.
Nature Protocols ( IF 14.8 ) Pub Date : 2017-Nov-01 , DOI: 10.1038/nprot.2017.049
Suman Kumar Maity , Muhammad Jbara , Guy Mann , Guy Kamnesky , Ashraf Brik

Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl]2 and PdCl2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within ∼20-30 d.

中文翻译:

组蛋白及其类似物的全化学合成,辅以天然化学连接和钯配合物。

组蛋白的化学合成可以通过衍生氨基酸的偶联来精确控制翻译后修饰的安装。获得用于表观遗传研究的修饰的组蛋白的其他方法的缺点包括所获得的产物的异质性和难以在同一组蛋白上掺入多种修饰的困难。在该协议中,未保护的肽片段通过Fmoc固相合成制备,并通过天然化学连接(NCL)在水性缓冲液中偶联;在NCL中,在具有N端Cys的肽与另一个具有C的肽之间形成了肽键-末端硫酯)。该任务具有挑战性,与疏水肽的制备和连接有关的障碍,以及由于多肽组装过程中的保护基操作而需要进行多个纯化步骤。为了解决这个问题,我们的方法使用易于去除的增溶标签进行疏水肽的合成和连接,以及使用钯化学方法(特别是[Pd(allyl)Cl ]2和PdCl 2复合物)。这种方法的效用在Lys34处泛素化的H2B,Tyr57处的磷酸化H2A和未修饰的H4的合成中得到了证明。这些类似物中的每一个都可以在约20至30 d内以毫克量制备。
更新日期:2017-10-11
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