Abstract
The 6xHis/Ni-NTA system is a fast and versatile tool for the affinity purification of recombinant proteins and antigenic peptides. It is based on the high-affinity binding of six consecutive histidine residues (the 6xHis tag) to immobilized nickel ions, giving a highly selective interaction that allows purification of tagged proteins or protein complexes from <1% to >95% homogeneity in just one step (1, 2). The tight association between the tag and the resin allows contaminants to be easily washed away under stringent conditions, yet the bound proteins can be gently eluted by competition with imidazole, or a slight reduction in pH. Moreover, because the interaction is independent of the tertiary structure of the tag, 6xHis labeled proteins can be purified even under the strongly denaturing conditions required to solubilize inclusion bodies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Further Reading Reviews
Hochuli, E. (1990) Purification of recombinant proteins with metal chelate adsorbent. Gene. Engineer., vol. 12 (Setlow, J. K., ed.), Plenum, New York, pp. 87–98.
Selected Examples
Abate, C., Luk, D., Gentz, R., Rauscherin, F. J., and Curran, T. (1990) Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly. Proc. Natl. Acad. Sci. USA 87, 1032–1036.
Bush, G. L., Tassin, A., Friden, H., and Meyer, D. I. (1991) Purification of a translocation-competent secretory protein precursor using nickel ion affinity chromatography. J. Biol. Chem. 266, 13,811–13,814.
Gentz, R., Certa, U., Takacs, B. J., Matile, H., Dobeli, H., Pink, R., Mackay, M., Bone, N., and Scaife, J. G. (1988) Major surface antigen pl90 of Plasmo-dium falciparum: detection of common epitopes present in a variety of Plas-modia isolates. EMBO J. 7, 225–230.
Gentz, R., Chen, C, and Rosen, C. A. (1989) Bioassay for trans-activation using purified immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis. Proc. Natl. Acad. Sci. USA 86, 821–824.
Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R., and Stuber, D. (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology 6, 1321–1325
Le Grice, S. F. J. and Grueninger-Leitch, F. (1990) Rapid purification of homodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur. J. Biochem. 187, 307–314
Stuber, D., Bannwarth, W., Pink, J. R. L., Meloen, R. H., and Matile, H. (1990) New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein. Eur. J. Immunol. 20, 819–824.
Takacs, B. J. and Girard, M.-F. (1991) Preparation of clinical grade proteins produced by recombinant DNA technologies. J. Immunol. Meth. 143, 231–240.
References
Stilber, D., Matile, H., and Garotta, G. (1990) System for high-level production in Escherichia coli and rapid purification of recombinant proteins: application toepitope mapping, preparation of antibodies, and structure-function analysis, in Immunological Methods (Lefkovits, I. and Pernis, B., eds.), vol. IV, Academic, New York, pp. 121–152.
Janknecht, R., de Martynoff, G., Lou, J., Hipskind, R. A., Nordheim, A., and Stunnenberg, H. G. (1991) Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc. Natl. Acad. Sci. USA 88, 8972–8976.
Dobeli, H., Trecziak, A., Gillessen, D., Matile, H., Srivastava, I. K., Perrin, L. H., Jakob, P. E., and Certa, U. (1990) Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase. Molec. Biochem. Parasitol. 41, 259–268.
Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598–599
Sulkowski, E. (1985) Purification of proteins by IMAC. Trends Biotechnol. 3, 1–7.
Hochuli, E., Ddbeli, H., and Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatog. 411, 177–184.
Takacs, B. J. (1979) in Immunological Methods (Lefkovits, I. and Pernis, B., eds.), vol. 1, Academic, New York, p 81.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1994 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stiiber, D., Henco, K. (1994). 6xffis-Ni-NTA Chromatography as a Superior Technique in Recombinant Protein Expression/Purification. In: Harwood, A.J. (eds) Protocols for Gene Analysis. Methods in Molecular Biology, vol 31. Humana Press. https://doi.org/10.1385/0-89603-258-2:371
Download citation
DOI: https://doi.org/10.1385/0-89603-258-2:371
Publisher Name: Humana Press
Print ISBN: 978-0-89603-258-3
Online ISBN: 978-1-59259-518-1
eBook Packages: Springer Protocols