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.
Key WordsRNA editing RNA-binding protein affinity chromatography Apobec-1 complementation factor