Purification and Assay of Recombinant ADAR Proteins Expressed in the Yeast Pichia pastoris or in Escherichia coli

ADARs are found in Metazoans but are not present in yeasts. We have found that the methanol-utilizing yeast
Pichia pastoris
can be used to efficiently express enzymatically active epitope-tagged ADARs. We describe plasmid construction and protein expression procedures for producing
Drosophila
ADAR in this system.
ADAR expression in
Pichia pastoris
uses the methanol-inducible alcohol oxidase
AOX1
promoter for induction. A Zeocin resistance gene on the plasmid is used to select high copy number tandem integrations of the plasmid constructs. Preparation of extracts by grinding cultures in liquid nitrogen and purification protocols using 6 � HIS and FLAG epitope tags are described. Procedures for preparing radiolabeled dsRNA and for assaying the non-specific RNA editing activity of ADARs are described.ADARs produced in
Escherichia coli
are not enzymatically active. We describe expression of the ADAR dsRNA binding domains in
E. coli
using current versions of the T7 promoter based Studier vectors as well as the purification of the domains.