Immobilized metal chelate chromatography

Patwardhan, A.V. and Ataai, M.M., Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on Chelating Superose, /. Chromatogr. A, 767, 11, 1997. 

For identification and purification, recombinant proteins are often tagged to the N-terminus with an additional sequence of histidyl residues, mostiysix (Hisg tag). This tag binds selectively to cations as nickel or copper immobilized by covalent chelators as nitrilotri-acetic acid. The method is named Metal Chelate Chromatography (MCC, MCAC, IMAC). 

In 1970s, first application of metal-chelate affinity chromatography which is later named as "immobilized-metal (ion) affinity chromatography (IMAC) was perfomed. Metal-chelate chromatography technique exploits selective interactions and affinity between transition metal immobilized on a solid support (resin) via a metal chelator and amino acid residues which act as electron donors in the protein of interest [25-26]. As well as aromatic and heterocyclic compounds, proteins such as histidine, tyrosine, tyriptophane and phenylalanine posses affinity to transition metals which form complexes with compounds rich in electrons [25,27]. 

Several scenarios can be considered with immobilized metal affinity chromatography (IMAC) systems in terms of the relative influences of these different equilibria. Thus, if Ksolvate > Kpj, the polypeptide or protein will not bind to the IMAC sorbent. Similarly, if KPl > KM + w CheL the polypeptide or protein will strip the metal ion from the immobilized chelating complex, such as observed, e.g., with native or recombinant fibroblast growth... 

This type of affinity separation is also known under the name of immobilized metal affinity chromatography (IMAC) and is based on the ion-mediated interaction with proteins. Metal ions are adsorbed first on a chelating... 

Immobilized metal affinity chromatography has been shown to be effective for isolating proteins from crude mixtures, as well as for selective separations of closely related proteins [2]. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography and the immobilized metalion complexes are much more robust than antibodies or enzymes. These factors make IMAC particularly well suited for scale-up to process scale chromatography. The main scale-up points to be aware of are the degree to which the column is metal saturated, the chelating agent content of the sample, and the potential of leached metal (or its interactions) within the product eluate. 

Burba, P., Jakubowski, B., Kuckuk, R., Kiillmer, K., and Heumann, K. G., Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron (lll)-loaded ion exchangers, Fresenius J. Anal Chem., 368, 689-696, 2000. 

Immobilized-metal affinity chromatography (IMAC) is a separation technique that uses covalently bound chelating compounds on solid chromatographic supports to entrap metal... 

Hynek, R. Kozak, A. Drab, V. Sajdok, J. Kas, J. Charecterization of Casein phosphopeptides in cheese extracts using a combination of immobilized metal chelate affinity and leversed-phase high-performance liquid chromatography. Adv. Food Sci. (CMTL) 1999, 21 (5/6), 192. 

Zacharion, M. Traverso, I. Hearn, M.T.W. High-performance liquid chromatography of amino acids, peptides and proteins CXXXI. 0-Phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins. J. Chromatogr. 1993, 646, 107-120. 

Immobilized Metal Affinity Chromatography. The buffers used in the initial His-tag purification are detailed in Table II. The 50 mL lysate is loaded onto a 1 mL NP -charged HiTrap Chelating HP column (Amersham Pharmacia, Cat. 17-0408-01) nsing the AKTA FPLC system with Frac-950 fraction collector (Amersham Pharmacia). The pnrification scheme is detailed in Table III. The His-tagged Sec23/24 complex should elute at an Imidizole concentration aronnd 150 mM with a typical chromatogram and resultant western blot of fractions shown in Fig. 2. 

Metal interaction chromatography is an HPLC technique that can separate many biopolymers because of their differential ability to form complexes with metal ions [1]. It employs a stationary phase with an appropriate metal immobilized via chelating functions bound to the surface. Retention and separation of the sample components occur largely by their interaction with the chelated metal. Although immobilized metal affinity chromatography (IMAC) is a common name for the technique, it is called metal interaction chromatography (MIC) in this book to conform to the nomenclature used for the other interactive chromatographic methods for biopolymer separation by HPLC. 

Another form of affinity chromatography that is applied in separation of protein is immobilized metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC). In IMAC, metal ions are affixed to adsorbents by chelating agents such as iminodiacetic acid (IDA), and tris (carboxymethyl)-ethylenediamine (TED) via a long hydrophilic spacer arm. The principle behind IMAC is that many transition metal ions like zinc and copper can coordinate to amino acids such as histidine, cysteine and tryptophan via electron donor groups on the amino acid side chain [8]. 

Particularly, immobilized-metal affinity chromatography (IMAC) is a separation technique that uses covalently bound chelating compounds on solid chromatographic supports to chelate metal ions, which serve as affinity ligands for various proteins, making use of coordinative binding of some amino acid residues exposed on the surface. Most commonly used are Cu(II), Ni(II), Zn(II), Co(II), and Fe(III) transition-metal ions, which are electron-pair acceptors and can be considered as Lewis acids. MAC technique can be used to separate proteins and peptides, with exposed histidine residues, which are primarily responsible for binding to immobilized metal ions. 

Epoxy resins with alternative spacer group chemistries have been used for the preparation of resins for immobilized metal-chelate affinity chromatography (IMAC). The following materials have been synthesized [56] ... 

Immobilized metal-affinity chromatography (IMAC) is also known as metal-chelate affinity chromatography (MCAC). This method was first proposed by Porath et al. in 1975 [63] and is based on the specific interactions between immobilized metal ions and amino acid residues, such as histidine, fiyptophan, and cysteine in proteins or peptides [63]. IMAC has become an important tool for the detection and purification of metalloproteins, histidine-tagged proteins, and phosphorylated proteins. Areas in which this method is now used include proteomics [64—66], work with recombinant proteins [67—69], and disease diagnosis [70,71]. 

Metal-chelate affinity chromatography is a powerful purification technique whereby proteins or other molecules can be separated based upon their ability to form coordination complexes with immobilized metal ions (Porath et al., 1975 Lonnerdal and Keen, 1982 Porath and Belew, 1983 Porath and Olin, 1983 Sulkowski, 1985 Kagedal, 1989). The metal ions are stabilized on a matrix through the use of chelating compounds which usually have multivalent points of interaction with the metal atoms. To form useful affinity supports, these metal ion complexes must have some free or weakly associated and exchangeable coordination sites. These exchangeable sites then can form complexes with coordination sites on proteins or other molecules. Substances that are able to interact with the immobilized metals will bind and be retained on... 

Figure 6.17 Schematic representation of the basic principles of metal chelate affinity chromatography. Certain proteins are retained on the column via the formation of coordinate bonds with the immobilized metal ion (a). The actual structure of the most commonly used metal chelator, iminodiacetic acid, is presented in (b)...

Metal-chelate affinity chromatography (Immobilized-metal (Ion) affinity chromatography)... 

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