Allerson Charles R, Martinez Alan, Yikilmaz Emine, Rouault Tracey A
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
RNA. 2003 Mar;9(3):364-74. doi: 10.1261/rna.2143303.
Regulated expression of proteins involved in mammalian iron metabolism is achieved in part through the interaction of the iron regulatory proteins IRP1 and IRP2 with highly conserved RNA stem-loop structures, known as iron-responsive elements (IREs), that are located within the 5' or 3' untranslated regions of regulated transcripts. As part of an effort to determine the structures of the IRP-IRE complexes using crystallographic methods, we have developed an efficient process for obtaining functionally pure IRP1 and IRP2 that relies upon the improved overexpression (>10 mg of soluble IRP per liter of culture) of each human IRP in the yeast Pichia pastoris and large-scale purification using RNA affinity chromatography. Despite the utility of RNA affinity chromatography in the isolation of RNA-binding proteins, current methods for preparing RNA affinity matrices produce columns of low capacity and limited stability. To address these limitations, we have devised a simple method for preparing stable, reusable, high-capacity RNA affinity columns. This method utilizes a bifunctional linker to covalently join a 5'-amino tethered RNA with a thiol-modified Sepharose, and can be used to load 150 nmole or more of RNA per milliliter of solid support. We demonstrate here the use of an IRE affinity column in the large-scale purification of IRP1 and IRP2, and suggest that the convenience of this approach will prove attractive in the analysis of other RNA-binding proteins.
哺乳动物铁代谢相关蛋白的表达调控部分是通过铁调节蛋白IRP1和IRP2与高度保守的RNA茎环结构（称为铁反应元件，IREs）相互作用实现的，这些结构位于受调控转录本的5'或3'非翻译区内。作为使用晶体学方法确定IRP - IRE复合物结构工作的一部分，我们开发了一种高效的方法来获得功能纯的IRP1和IRP2，该方法依赖于在毕赤酵母中提高每个人类IRP的过表达水平（每升培养物中可溶性IRP超过10毫克），并使用RNA亲和色谱进行大规模纯化。尽管RNA亲和色谱在分离RNA结合蛋白方面很有用，但目前制备RNA亲和基质的方法产生的柱子容量低且稳定性有限。为了解决这些限制，我们设计了一种制备稳定、可重复使用、高容量RNA亲和柱的简单方法。该方法利用双功能接头将5'-氨基连接的RNA与硫醇修饰的琼脂糖共价连接，每毫升固体支持物可负载150纳摩尔或更多的RNA。我们在此展示了IRE亲和柱在大规模纯化IRP1和IRP2中的应用，并表明这种方法的便利性在分析其他RNA结合蛋白时将具有吸引力。
