{"title":"Purification of Recombinant Histones and Mononucleosome Assembly","authors":"Yiming Zhao, Jingjun Hong","doi":"10.1002/cpz1.70155","DOIUrl":null,"url":null,"abstract":"<p>The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a <span>histone octamer</span>. Each histone octamer is composed of two copies each of the histone proteins <span>H2A</span>, <span>H2B</span>, <span>H3</span>, and <span>H4</span>. Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from <i>Escherichia coli (E. coli)</i>, their subsequent reconstitution as octamers, and assembly into mononucleosomes <i>in vitro</i>. Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Purification of recombinant histones and mononucleosome assembly</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2000,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.70155","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from Escherichia coli (E. coli), their subsequent reconstitution as octamers, and assembly into mononucleosomes in vitro. Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC.
Basic Protocol: Purification of recombinant histones and mononucleosome assembly
重组组蛋白纯化及单核小体组装
核小体是染色质的基本单位。每个核小体由不到两圈的DNA组成，这些DNA包裹着一组叫做组蛋白的八种蛋白质，这些蛋白质被称为组蛋白八聚体。每个组蛋白八聚体由组蛋白H2A、H2B、H3和H4的两个拷贝组成。核小体在基因表达和调控中起着重要作用。而之前的方案使用HPLC（高效液相色谱法）纯化每个组蛋白，在八聚体重构后，随后使用FPLC（快速蛋白液相色谱法）纯化八聚体。本文提出了一种仅用FPLC进行八聚体重组和单核小体组装的方法。本基本方案描述了从大肠杆菌（E. coli）中纯化组蛋白的程序，随后将其重组为八聚体，并在体外组装成单核小体。通过这种方法，可以在不使用高效液相色谱的情况下大规模地重组组蛋白八聚体和单核小体。总的来说，该协议描述了一个有效的新方法，重建八聚体和组装的单核小体。©2025 Wiley期刊公司。基本方案：重组组蛋白和单核小体组装的纯化
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