Isolation of an mRNA-Binding Protein Involved in C-to-U Editing

This chapter describes the technique of RNA affinity chromatography, which is a powerful approach for isolating RNA-binding proteins. This method takes advantage of the fact that sequence-specific RNA-binding proteins often bind their targets with high affinity. Here we outline a protocol for purifying Apobec-1 complementation factor (ACF), the RNA-binding subunit of the apolipoprotein-B (apo-B) mRNA-editing enzyme. ACF was purified using synthetic wild-type and mutant apo-B RNAs, which were coupled to cyanogen bromide (CNBr)-activated Sepharose. The methods are plasmid construction for in vitro transcription, affinity chromatography column preparation, protein purification by RNA affinity chromatography, and analysis of the purified protein.