Abstract
This protocol describes affinity purification of bacterially expressed, recombinant membrane proteins fused with calmodulin-binding domains. As exemplified by the Escherichia coli nicotinamide nucleotide transhydrogenase, this method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA and, unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). Our protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Stofko-Hahn, R.E., Carr, D.W. & Scott, J.D. A single step purification for recombinant proteins. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase. FEBS Lett. 302, 274–278 (1992).
Zheng, C.F., Simcox, T., Xu, L. & Vaillancourt, P. A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins. Gene 186, 55–60 (1997).
Wyborski, D.L., Bauer, J.C., Zheng, C.F., Felts, K. & Vaillancourt, P. An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions. Protein Expr. Purif. 16, 1–10 (1999).
Klein, W. Calmodulin-binding peptide as a removable affinity tag for protein purification. Methods Mol. Biol. 205, 79–97 (2003).
Vaillancourt, P., Zheng, C.F., Hoang, D.Q. & Breister, L. Affinity purification of recombinant proteins fused to calmodulin or to calmodulin-binding peptides. Meth. Enzymol. 326, 340–362 (2000).
Egorov, M.V. et al. Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+-ATPase. Protein Expr. Purif. 36, 31–39 (2004).
Niggli, V., Penniston, J.T. & Carafoli, E. Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J. Biol. Chem. 254, 9955–9958 (1979).
Penniston, J.T., Filoteo, A.G., McDonough, C.S. & Carafoli, E. Purification, reconstitution, and regulation of plasma membrane Ca2+-pumps. Meth. Enzymol. 157, 340–351 (1988).
Ishii, T. & Takeyasu, K. The C-terminal 165 amino acids of the plasma membrane Ca2+-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K+-ATPase α-subunit. EMBO J. 14, 58–67 (1995).
Acknowledgements
The authors wish to thank T.I. Muravieva for the gift of crude calmodulin. This work was made possible in part by funding from the Swedish Natural Science Research Council, the Russian Foundation for Basic Research and INTAS.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Pestov, N., Rydström, J. Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase. Nat Protoc 2, 198–202 (2007). https://doi.org/10.1038/nprot.2006.456
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.456
This article is cited by
-
Direct purification and immobilization of his-tagged enzymes using unmodified nickel ferrite NiFe2O4 magnetic nanoparticles
Scientific Reports (2023)
-
Characterizing metal-binding sites in proteins with X-ray crystallography
Nature Protocols (2018)
-
Cloning and Expression of Brain-Specific Anion Transporter BSAT1 and Isolation of Monoclonal Antibodies to Its Extracellular Fragment
Bulletin of Experimental Biology and Medicine (2012)
-
Isolation of Extracellular Recombinant Fragment of Rat Connexin-43
Bulletin of Experimental Biology and Medicine (2009)
-
Immunofluorescent Analysis of Connexin-43 Using Monoclonal Antibodies to Its Extracellular Domain
Bulletin of Experimental Biology and Medicine (2009)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
