Expression and purification of recombinant human histones
Expression and purification of recombinant human histones
复制标题
DOI：
10.1016/j.ymeth.2003.10.024
复制
发表时间：
2004-05-01
期刊：
影响因子：
4.8
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
--
作者：
Tanaka, Y;Tawaramoto-Sasanuma, M;Yokoyama, S
从细菌表达的历史重构的核小体对于历史变体、组蛋白突变体和组蛋白翻译后修饰的功能和结构分析是有用的。在本研究中，我们建立了一种新的表达和纯化重组人组蛋白的方法。人类历史H2 A、H2 B和H3基因在大肠杆菌细胞中表达良好。而人组蛋白H4基因表达较弱。因此，我们设计了一个新的历史H4基因的密码子优化的大肠杆菌表达系统和构建的H4基因的化学合成的寡脱氧核苷酸。重组人的历史表达为六组氨酸标记的蛋白质，并通过一步色谱法与镍-次氮基三乙酸琼脂糖在6 M尿素的存在下进行纯化。H2 A/H2 B二聚体和H3/H4四聚体通过对不含尿素的缓冲液透析进行重折叠，并且通过凝血酶蛋白酶消化除去H2 A/H2 B二聚体和H3/H4四聚体中的历史的六组氨酸标签。通过盐透析法证实了通过该方法获得的H2 A/H2 B二聚体和H3/H4四聚体精通核小体形成。人类CENP-A基因是着丝粒特异性组蛋白H3变体，包含28个大肠杆菌次要密码子。杆菌一个新的CENP-A基因为E.构建了大肠杆菌表达系统。纯化的重组CENP-A蛋白与组蛋白H2 A、H2 B形成核小体样结构。还有H4。(C)2003年爱思唯尔公司All rights reserved.
Nucleosomes reconstituted from bacterially expressed histories are useful for functional and structural analyses of historic variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human historic H2A, H2B, and H3 genes were expressed well in Escherichia coli cells. but the human histone H4 gene was poorly expressed. Therefore, we designed a new historic H4 gene with codons optimized for the E coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histories were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histories in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed. and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B. and H4. (C) 2003 Elsevier Inc. All rights reserved.