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

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


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 


  1. 1.
    Keegan, L. P., Gallo, A., and O’Connell, M. A. (2001) The many roles of an RNA editor. Nat. Rev. Genet. 2(11), 869–878.PubMedCrossRefGoogle Scholar
  2. 2.
    O’Connell, M. A., Gerber, A., and Keegan, L. P. (1998) Purification of native and recombinant double-stranded RNA-specific adenosine deaminases. Methods 15, 51–62.CrossRefGoogle Scholar
  3. 3.
    Ohman, M., Kallman, A. M., and Bass, B. L. (2000) In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site. RNA 6(5), 687–697.PubMedCrossRefGoogle Scholar
  4. 4.
    Lai, F., Chen, C. X., Lee, V. M., and Nishikura, K. (1997) Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases. Mol. Cell. Biol. 17, 2413–2424.PubMedGoogle Scholar
  5. 5.
    Herbert, A., Wagner, S., and Nickerson, J. A. (2002) Induction of protein translation by ADAR1 within living cell nuclei is not dependent on RNA editing. Mol. Cell 10(5), 1235–1246.PubMedCrossRefGoogle Scholar
  6. 6.
    Palladino, M. J., Keegan, L. P., O’Connell, M. A., and Reenan, R. A. (2000) dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing. RNA 6, 1004–1018.PubMedCrossRefGoogle Scholar
  7. 7.
    Cregg, J. M., Barringer, K. J., Hessler, A. Y., and MAdden, K. R. (!985) Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5(12), 3376–3385.Google Scholar
  8. 8.
    Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (NY) 11(8), 905–910.PubMedCrossRefGoogle Scholar
  9. 9.
    Wegner, G. H. (1990) Emerging applications of the methylotrophic yeasts. FEMS Microbiol Rev. 7(3-4), 279–283.PubMedGoogle Scholar
  10. 10.
    Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189(1), 113–130.PubMedCrossRefGoogle Scholar
  11. 11.
    Studier, F. W., et al. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89.PubMedCrossRefGoogle Scholar
  12. 12.
    Bass, B. L. and Weintraub, H., (1988) An unwinding activity that covalently modifies its double-strand RNA substrate. Cell 55, 1089–1098.PubMedCrossRefGoogle Scholar
  13. 13.
    O’Connell, M. A. and Keller, W., (1994) Purification and properties of double-stranded RNA-specific adenosine deaminase from calf thymus. Proc. Natl. Acad. Sci. USA 91, 10,596–10,600.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.MRC Human Genetics UnitWestern General HospitalEdinburghUK

Personalised recommendations