Name: generation and purification of human ino80 chromatin remodeling complexes and subcomplexes
Uploaded: 2014-10-23T13:00:00
Description: Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

在作者的实验室工作是由普通医学科学研究所（GM41628）的赠款和赠款的斯托尔斯医学研究所的海伦·纳尔逊医学研究基金，在大堪萨斯城社区基金会的支持。

1代和HEK293稳定细胞系表达全长或者标记的突变版本文化抗原标记INO80或其他INO80复杂的亚基<ol><li>克隆的cDNA编码全长或突变的人INO80 ATP酶或另一INO80亚基入哺乳动物表达载体pcDNA5 / FRT用在帧中，N末端FLAG表位标签。 </li><li>出发前请确认所插入的cDNA通过DNA测序的序列。 </li><li>执行转染，成长的Flp-在HEK293细胞中，10厘米的组织培养皿在含有DMEM（Dulbecco改良的Eagle培养基），5％的谷氨酰胺的培养基中?...

<ol>
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从高等真核生物多亚基哺乳类染色质重塑复合物的结构和功能的研究已经受到阻碍制备含有突变亚基或完全缺乏某些亚基这种配合物的生物化学有用量的难度。有许多技术障碍：第一，基因操作在哺乳动物细胞中已经在技术上具有挑战性的并且耗时的。不同于酵母细胞，其基因组可以容易地编辑和定向用重组工程技术，在哺乳动物基因组中是更复杂的结构和较少受重组工程干预。因此，缺失或哺?...

进化上保守的SNF2家庭染色质重塑复合物染色质组织和DNA可达1的主要监管机构。这些重塑复合物总是包括中央SNF2状ATP酶亚基，其中，在某些情况下，装配有各种辅助蛋白，并形成多亚基大分子装配体。研究ATP依赖的染色质重塑过程的分子细节，以理解亚基和/或结构域的结构的特定子集的贡献，以复合物的活性是很重要的。这样的分析要求，以产生缺乏特定蛋白质亚基或结构域的结构高度纯化的突变体复合物的能力。 的依赖ATP的染色质重塑复合物之前的结构-功能研究已经广泛地聚焦在酵母模型系统上，由于酵母基因组的优异的可操作性（参见，例如，文献1-4）。由于养护其中同源重塑复合物亚基组成和功能，结构和酵母重塑复合物的功能研究高等真核生物提供了重要的见解的同行。然而，明显的种属特异性重塑复合物之间的差异确实存在，从增益或物种特异性的亚基，增益或保守的亚单位的种特异性结构域的损失，和序列变异性的保守亚基的保守结构域之内的损失所导致。这样的差异可以在原则上需要高等真核细胞，以适应新的分子和细胞的环境中进行驱动。因此，理解的高等真核质重塑复合物亚基如何向核小体重构过程是有价值的，因为它不仅揭示了ATP依赖的染色质重塑过程的基本机制的光，而且还可以提供有价值的洞察机制，其中的染色质结构在集锦和基因表达她真核生物规管。 到目前为止，已经出现了只有有限的多亚基哺乳动物染色质重塑复合物的结构和功能的研究，部分是由于难以获得生化定义染色质重塑复合物及其亚。我们已经部分地规避这些困难与下面描述的方法，其中免疫亲和纯化用于制备从人体细胞完好INO80或INO80 subcomplexes稳定表达N末端​​FLAG表位标记的野生型或INO80 5-7的突变版本（ 图1） 。以获得从人体细胞完好INO80复合物，FLP介导的重组被用于产生转基因的HEK293细胞系中稳定表达FLAG表位标签的编码cDNA的INO80复合物8-10的亚单位组成。因为过度表达INO80亚基可以是有些有毒，有必要分离并保持下选择性共克隆细胞系条件，以确保在需要扩张大规模细胞培养物的多通道的稳定的转基因表达。要获得含有亚单位的一个子集较小INO80 subcomplexes，我们已经成功地使用两种方法（ 图2A，B）。在所述第一，我们生成的HEK293 FLP-在细胞系中稳定表达INO80的突变版本，缺乏具体的亚基5所需的相互作用结构域。或者，siRNA的介导击倒用于消耗从细胞中表达适当的FLAG标记的INO80亚基（未发表的数据）所需的子单元。最后，为了净化人类INO80复合物，FLAG琼脂糖基于色谱11被用于从核提取物丰富的含INO80馏分，从而有效地减少了污染的胞质蛋白在含有纯化INO80或INO80 subcomplexes的最后部分的存在。

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</table>

generation and purification of human ino80 chromatin remodeling complexes and subcomplexes

INO80染色质重塑复合物通过催化ATP依赖的核小体重塑核监管动态和DNA的可访问性。人类INO80物包括14蛋白亚基包括INO80一个SNF2般的ATP酶，它同时作为催化亚基和支架的复合物的组装。另一亚基的功能和由此来的INO80复合物的染色质重塑活性它们贡献的机制仍然知之甚少，部分原因是由于产生INO80组件在人细胞或异源表达系统的挑战。这JOVE协议描述的程序，可以让那些缺乏亚基或亚基的一个子集人类INO80染色质重塑subcomplexes的净化。 N末端FLAG表位标签的INO80基因被稳定地导入人胚胎肾（HEK）用FLP-介导的重组293细胞系。在该INO80复合物的亚基的一个子集是B中的事件ê删除，1表示是缺乏必要对这些亚基组装的平台，而不是突变INO80蛋白质。在该事件的个体亚基是被耗尽的，针对这一亚基成的HEK 293细胞系的稳定表达FLAG 1 transfects的siRNA标记INO80 ATP酶。核提取物的制备和免疫标志进行充实含INO80衍生蛋白组分。纯化INO80 subcomplexes的组合物然后可以使用的方法，例如免疫印迹，银染色法和质谱法进行分析。产生的INO80和INO80 subcomplexes根据该协议可以使用各种生化测定法，其中在附图JOVE协议描述来进一步分析。这里描述的方法可以适用于任何哺乳动物多亚基染色质重塑的结构和功能特性的研究和改进的配合物。

图1示出的流程图总结了用于产生，纯化和表征人类INO80 ATP依赖的染色质重塑复合物的过程。 如示于图2和3中 ，这些程序使缺乏各种亚基，从而使这些缺失亚基INO80的酶活性的贡献后续的生化分析野生型INO80和INO80 subcomplexes的产生， 图2描述了两种策略我们用于定义INO80复合物的结构和生成INO80 subcomplexes。在第一阶段， ?...

Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

这个协议描述了用于产生和纯化野生型和人INO80染色质重构复合物的突变版本中的一个过程。表位标记的INO80亚基的版本都稳定地表达在HEK293细胞中，并完全配合物和配合物缺乏亚基的特定集合是通过免疫亲和层析纯化。

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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