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

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 265)

Abstract

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.

Key Words

ADAR RNA-editing RNA interference deaminase dsRNA Pichia pastoris Drosophila melanogaster Escherichia coli protein overproduction protein purification ion channel nervous system 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.MRC Human Genetics UnitWestern General HospitalEdinburghUK

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