A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris.
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.
Allerson
Charles R
CR
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Martinez
Alan
A
Yikilmaz
Emine
E
Rouault
Tracey A
TA
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D035941:Iron Regulatory Protein 1 / Q000302:isolation & purification* / Q000378:metabolism; D035942:Iron Regulatory Protein 2 / Q000302:isolation & purification* / Q000378:metabolism; D010843:Pichia; D030821:Plants, Genetically Modified; D012313:RNA / Q000378:metabolism*
D012313:RNA; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2