Immobilized Metal-Ion Affinity Chromatography
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Immobilized metal-ion affinity chromatography (IMAC) (1–4) is also referred to as metal chelate chromatography, metal-ion interaction chromatography, and ligand-exchange chromatography. We view this affinity-separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider-spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., refs. 5–9) and on protein surfaces (10–15). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (17,18) is being explored continuously 19). A common fusion protein is the hexahistidine tag for purification (20). Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1.
Suitable metal ions
Commercial source a
Pharmacia LKB Pierce Sigma Boehringer-Mannheim TosoHaas
α-Alkyl nitrilotriacetic acid
Carboxymethylated aspartic acid
(GHHPH) n G b
- Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598–599. CrossRef
- Porath, J. and Olin, B. (1983) Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22, 1621–1630. CrossRef
- Garberc-Porekar, V. and Menart V. (2001) Perspectives of immobilized-metal affinity chromatography. J. Biochem. Biophys. Methods 49, 335–360. CrossRef
- Chaga, G S. (2001) Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J. Biochem. Biophys. Methods 49, 313–334. CrossRef
- Monjon, B. and Solms, J. (1987) Group separation of peptides by ligand-exchange chromatography with a Sephadex containing N-(2-pyridyl-methyl)glycine. Anal. Biochem. 160, 88–97. CrossRef
- Hochuli, E., Dobeli, H., and Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177–184. CrossRef
- Yip, T.-T. and Hutchens T. W. (1989) Development of high-performance immobilized metal affinity chromatography for the separation of synthetic peptides and proteolytic digestion products, in Protein Recognition of Immobilized Ligands (Hutchens, T. W., ed.), UCLA Symposia on Molecular and Cellular Biology Vol. 80, Alan R. Liss, New York, pp. 45–56.
- Yip, T. T., Nakagawa, Y, and Porath, J. (1989) Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography. Anal. Biochem. 183, 159–171. CrossRef
- Hutchens, T. W. and Yip, T. T. (1990) Differential interaction of peptides and protein surface structures with free metal ions and surface-immobilized metal ions. J. Chromatogr. 500, 531–542. CrossRef
- Sulkowski, E. (1985) Purification of proteins by IMAC. Trends Biotechnol 3, 1–7. CrossRef
- Hutchens, T. W. and Li, C. M. (1988) Estrogen receptor interaction with immobilized metals: differential molecular recognition of Zn2+, Cu2+, and Ni2+ and separation of receptor isoforms. J. Mol. Recogn. 1, 80–92. CrossRef
- Hutchens, T. W., Li, C. M., Sato, Y, and Yip, T.-T. (1989) Multiple DNA-binding estrogen receptor forms resolved by interaction with immobilized metal ions. Identification of a metal-binding domain. J. Biol. Chem. 264, 17,206–17,212.
- Hemdan, E. S., Zhao, Y.-J., Sulkowski, E., and Porath, J. (1989) Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA 86, 1811–1815. CrossRef
- Hutchens T W. and Yip, T.-T. (1991) Metal ligand-induced alterations in the surface structures of lactoferrin and transferrin probed by interaction with immobilized Cu(II) ions. J. Chromatogr. 536, 1–15. CrossRef
- Mantovaara-Jonsson, T, Pertoft, H., and Porath, J. (1989) Purification of human serum amyloid ccmponent (SAP) by calcium affinity chromatography. Biotechnol. Appl. Biochem. 11, 564–571.
- Fiedler, M. and Skerra, A. (2001) Purification and characterization of His-tagged antibody fragments. in Antibody Engineering (Kontermann R., ed) Springer-Verlag, Berlin, pp. 243–256.
- Hutchens, T. W., Nelson, R. W., Li, C. M., and Yip, T.-T. (1992) Synthetic metal binding protein surface domains for metal ion-dependent interaction chromatography. I. Analysis of bound metal ions by matrix-assisted UV laser desorption time-of-flight mass spectrometry. J. Chromatogr. 604, 125–132. CrossRef
- Hutchens, T. W. and Yip, T.-T. (1992) Synthetic metal binding protein surface domains for metal ion-dependent interaction chromatography. II. Immobilization of synthetic metal-binding peptides from metal-ion transport proteins as model bioactive protein surface domains. J. Chromatogr. 604, 133–141. CrossRef
- Hutchens, T W. and Yip, T.-T. (1990) Model protein surface domains for the investigation of metal ion-dependent macromolecular interactions and metal ion transfer. Methods 4, 79–96.
- Bornhorst, J. A. and Falke, J. J. (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 326, 245–254. CrossRef
- Hutchens, T W. and Yip, T.-T. (1990) Protein interactions with immobilized transition metal ions: quantitative evaluations of variations in affinity and binding capacity. Anal. Biochem. 191, 160–168. CrossRef
- Nakagawa, Y, Yip, T T, Belew, M., and Porath, J. (1988) High performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides. Anal. Biochem. 168, 75–81. CrossRef
- Fatiadi A. J. (1987) Affinity chromatography and metal chelate affinity chromatography. CRC Crit. Rev. Anal. Chem. 18, 1–44.
- Kagedal, L. (1989) Immobilized metal ion affinity chromatography, in High Resolution Protein Purification (Ryden, L. and Jansson, J.-C, eds.), Verlag Chemie, Deerfield Beach, FL, pp. 227–251.
- Muszynska, G., Zheo., Y.-J., and Porath, J. (1986) Carboxypeptidase A: a model for studying the interaction of proteins with immobilized metal ions. J. Inorg. Biochem. 26, 127–135. CrossRef
- Holmes, L. D. and Schiller, M. R. (1997) Immobilized iron (III) metal affinity chromatography for the separation ogf phophorylated macromolecules: ligands and applications. J. Liquid Chromatogr. 20, 123–142. CrossRef
- Andersson, L. (1996) The use of immobilized Fe3+ and other hard metal ions in chromatography of peptides and proteins. Int. J. Biochromatogr. 2, 25–36.
- Yip, T.-T. and Hutchens, T W. (1991) Metal ion affinity adsorption of a ZN(II)-transport protein present in maternal plasma during lactation: structural characterization and identification as histidine-rich glycoprotein. Protein Express. Purif. 2, 355–362. CrossRef
- Hutchens, T W., Nelson, R. W., and Yip, T.-T. (1992) Recognition of transition metal ions by peptides: identification of specific metal-binding peptides in proteolytic digest maps by UV laster desorption time-of-flight spectrometry. FEBS Lett. 296, 99–102. CrossRef
- Hutchens, T W., Nelson, R. W., and Yip, T.-T. (1991) Evaluation of peptide-metal ion interactions by UV laser desorption time-of-flight mass spectrometry. J. Mol. Recogn. 4, 151–153. CrossRef
- Hutchens, T W., Nelson, R. W., Allen, M. H., Li, C. M., and Yip, T.-T. (1992) Peptide metal ion interactions in solution: detection by laser desorption time-of-flight mass spectrometry and electrospray ionization mass spectrometry. Biol. Mass Spectrom. 21, 151–159. CrossRef
- Hutchens, T W. and Yip, T.-T. (1991) Protein interactions with surface-immobilized metal ions: structure-dependent variations in affinity and binding capacity constant with temperature and urea concentration. J. Inorg. Biochem. 42, 105–118. CrossRef
- Figueoroa, A., Corradini, C., Feibush, B., and Karger, B. L. (1986) High-performance immobilized metal ion affinity chromatography of proteins on iminodiacetic acid silica-based bonded phases. J. Chromatogr. 371, 335–352. CrossRef
- Hutchens, T. W., Yip, T.-T., and Porath, J. (1988) Protein interaction with immobilized ligands. Quantitative analysis of equilibrium partition data and comparison with analytical affinity chromatographic data using immobilized metal ion adsorbents. Anal. Biochem. 170, 68–182. CrossRef
- Hutchens, T. W. and Li, C. M. (1990) Ligand-binding properties of estrogen receptor proteins after interaction with surface-immobilized Zn(II) ions: evidence for localized surface interactions and minimal conformational changes. J. Mol. Recogn. 3, 174–179. CrossRef
- Immobilized Metal-Ion Affinity Chromatography
- Book Title
- Protein Purification Protocols
- pp 179-190
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- Methods in Molecular Biology
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- Humana Press
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- Humana Press
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