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 x 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.
Ring
Gillian M
GM
MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom.
O'Connell
Mary A
MA
Keegan
Liam P
LP
D000243:Adenosine Deaminase / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017393:RNA Editing / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D015183:Restriction Mapping / Q000379:methods; D013379:Substrate Specificity
D018076:DNA, Complementary; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D000243:Adenosine Deaminase