Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

  • Silvia C. Locatelli-Hoops
  • Alexei A. YeliseevEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)


Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor.

Key words

His-tag Strep-tag Dual affinity tags Membrane protein CB2 receptor StrepTactin Ni-NTA 



This work was supported by the Intramural Research Program of the National Institute on Alcoholism and Alcohol Abuse, National Institutes of Health.


  1. 1.
    Block H et al (2009) Immobilized-metal affinity chromatography (IMAC): a review. Methods Enzymol 463:439–473PubMedCrossRefGoogle Scholar
  2. 2.
    Expression and purification of proteins using Strp-tag and/or 6xHistidine-tag (2011)
  3. 3.
    Yeliseev A, Zoubak L, Gawrisch K (2007) Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor. Protein Expr Purif 53(1):153–163PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
  5. 5.
    Knecht S et al (2009) Oligohis-tags: mechanisms of binding to Ni2 + -NTA surfaces. J Mol Recognit 22(4):270–279PubMedCrossRefGoogle Scholar
  6. 6.
  7. 7.
    Voss S, Skerra A (1997) Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification. Protein Eng 10(8):975–982PubMedCrossRefGoogle Scholar
  8. 8.
    Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8(8):1668–1674PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Kapust RB et al (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14(12): 993–1000PubMedCrossRefGoogle Scholar
  10. 10.
    Kapust RB, Routzahn KM, Waugh DS (2002) Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3). Protein Expr Purif 24(1):61–70PubMedCrossRefGoogle Scholar
  11. 11.
    Cherry S, Tropea, JE, Waugh DS. Expression and purification of His-TEV(S219V)-Arg. pur_histev.pdf
  12. 12.
    Yeliseev AA et al (2005) Expression of human peripheral cannabinoid receptor for structural studies. Protein Sci 14(10): 2638–2653PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Berger C et al (2010) Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation. Protein Expr Purif 70(2):236–247Google Scholar
  14. 14.
    Vukoti K et al (2012) Stabilization of functional recombinant cannabinoid receptor CB2 in detergent micelles and lipid bilayers. PLoS One 7(10):e46290Google Scholar
  15. 15.
    Kimura T et al (2012) Recombinant cannabinoid type 2 receptor in liposome model activates g protein in response to anionic lipid constituents. J Biol Chem 287(6):4076–4087Google Scholar
  16. 16.
    Krepkiy D, Gawrisch K, Yeliseev A (2007) Expression and purification of CB2 for NMR studies in micellar solution. Protein Pept Lett 14(10):1031–1037PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Silvia C. Locatelli-Hoops
    • 1
  • Alexei A. Yeliseev
    • 1
    Email author
  1. 1.Laboratory of Membrane Biochemistry and BiophysicsNational Institute on Alcohol Abuse and Alcoholism, National Institutes of HealthBethesdaUSA

Personalised recommendations