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ADAR editing enzymes are found in all multicellular animals and are conserved in sequence and protein organization. The number of ADAR genes differs between animals, ranging from three in mammals to one in Drosophila. ADAR is also alternatively spliced to generate isoforms that can differ significantly in enzymatic activity. Therefore, to study the enzyme in vitro, it is essential to have an easy and reliable method of expressing and purifying recombinant ADAR protein. To add to the complexity of RNA editing, the number of transcripts that are edited by ADARs differs in different organisms. In humans there is extensive editing of Alu sequences, whereas in invertebrates transcripts expressed in the central nervous system are edited and this editing increases during development. It is possible to quantify site-specific RNA editing by sequencing of clones derived from RT-PCR products. However, for routine assaying of an edited position within a particular transcript, this is both expensive and time consuming. Therefore, a nonradioactive method based on poison primer extension assay is an ideal alternative.

Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies.