Immobilized Metal-Ion Affinity Chromatography

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Abstract

Immobilized metal-ion affinity chromatography (IMAC) (14) 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. 59) and on protein surfaces (1015). 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.

Schematic illustration of several types of immobilized metal-chelating group, including iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH) n G (where n=1,2, 3, and 5. (From refs. 17 and 18.)
a Locations: Pharmacia LKB, Uppsala, Sweden; Pierce, Rockford, IL; Sigma, St. Louis, MO; Boeh-ringer-Mannheim, Mannheim, Germany; TosoHaas, Philadelphia, PA.
b Letters represent standard one-letter amino acid codes (G = glycine; H = histidine; P = proline). The number of internal repeat units is given by n (n = 1, 2, 3, and 5).
Table 1
Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal-Ion Affinity Chromatography
Chelating group
Suitable metal ions
Ref.
Commercial source a
IDA
Transitional
(1, 2)
Pharmacia LKB Pierce Sigma Boehringer-Mannheim TosoHaas
2-Hydroxy-3[N-(2-pyridylmethyl)glycine]propyl
Transitional
5
Not available
α-Alkyl nitrilotriacetic acid
Transitional
6
Not available
Carboxymethylated aspartic acid
Ca(II)
15
Not available
TED
Transitional
2
Not available
(GHHPH) n G b
Transitional
17, 18
Not available