Co-expression as a convenient method for the production and purification of core histones in bacteria
Co-expression as a convenient method for the production and purification of core histones in bacteria
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DOI：
10.1016/j.pep.2010.03.013
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发表时间：
2010-08-01
影响因子：
1.6
通讯作者：
Dutnall, Robert N.
中科院分区：
文献类型：
--
作者：
Anderson, Megan;Huh, Joon H.;Dutnall, Robert N.
共表达提供了一个重要的策略，生产多蛋白复合物的生化和生物物理研究。我们发现组蛋白H2A和H2B（来自酵母、鸡或果蝇）的共表达导致可溶性异二聚体H2AH2B复合物的产生。如果果蝇组蛋白H3和H4与组蛋白伴侣Asf1共表达，它们也可以作为可溶性（H3H4）（2）异源四聚体复合物产生。可溶性H2AH2B和（H3H4）（2）可以通过简单的色谱技术纯化，并且具有与内源性组蛋白相似的性质。我们的方法应促进组蛋白的生产，染色质结构和调控蛋白的研究，与组蛋白相互作用。我们描述了一个简单的策略，用于构建共表达质粒，基于T7 RNA聚合酶系统，这是适用于其他系统。它提供了几个优点，用于快速创建质粒以表达两种或更多种蛋白质，并用于测试蛋白质的不同组合，以获得最佳的复合物生产，溶解度或活性。(C)2010年爱思唯尔公司All rights reserved.
Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity. (C) 2010 Elsevier Inc. All rights reserved.