Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.     

Three-phase affinity partitioning of proteins.

Three-phase partitioning is an elegant way to separate proteins directly from even the large volumes of crude suspensions. It was found that interfacing it with a metal-affinity-based step makes the technique highly selective. As "proof of the concept," soybean trypsin inhibitor was purified 13-fold with 72% recovery. As recombinant proteins (via their polyhistidine tags) and many other naturally occurring proteins are often purified by immobilized metal ion affinity chromatography, the technique described here should prove valuable in purifying biotechnologically important proteins at a large scale.     

Purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography

Over the past 10 years, the baculovirus-insect cell system has become a powerful and versatile tool for the expression of a variety of heterologous proteins. In order to simplify separation of a cloned protein from the baculovirus-insect expression system, we have cloned a gene encoding for the protein of interest, a structural protein (VP2) of a strain (E/DEL) of infectious bursal disease virus (IBDV), with a metal ion binding site (His)(5) at its C-terminus. This chimeric protein (VP2H) has been expressed and one-step affinity purified with immobilized metal ions (Ni(+2)). With antigen capture-enzyme-linked immunosorbent assay (AC-ELISA), we determined that the conformation of this chimeric protein was no different from the recombinant wild-type VP2 protein. However, the two proteins (VP2 and VP2H) can be distinguished and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected immunologically following Western blotting. (c) 1994 John Wiley & Sons, Inc.     

Immobilized metal ion affinity chromatography

This article describes the technique of immobilized metal ion affinity chromatography (1MAC). The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The number of stationary phases that can be synthesized for efficient chclation of metal ions is unlimited, but the critical consideration is that there is enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. The versatility of IMAC is one of its greatest assets. An important contribution to the correct use of IMAC for protein purification is a simplified presentation of the various sample elution procedures.     

Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. (c) 1995 John Wiley & Sons, Inc.     

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

Modeling of non-ideal displacement separation in immobilized metal ion affinity chromatography

To predict complex behavior in protein displacement systems of immobilized metal ion affinity chromatography (IMAC), numerical simulation of non-linear chromatography was developed and compared with the ideal solution of the model. The theoretical and experimental results demonstrate that the IMAC model can be successfully employed in predicting induced mobile phase modifier gradients and complex behavior in metal affinity displacement chromatography. The solute movement analysis is able to predict the displacement separation well characterized by the intersections of the operation line under the induced mobile phase modifier and effective displacer concentrations.     

Study of human cord blood lymphocytes by immobilized metal ion affinity partitioning

The potential of immobilized metal ion affinity partitioning (IMAP) using dextran-PEG+PEG-IDA-M(II) systems to separate mononuclear cells from cord blood has been evaluated. The distribution of B cells, T cells, monocytes and hematopoietic stem cells between PEG and dextran phases was determined by flow cytometry with fluorochrome-labelled specific antibodies. Comparing these values with the post-Ficoll repartition resulted in the determination of enrichment factors, for each subpopulation, in the different phases. We were able to distinguish the partition pattern of B cells, T cells, monocytes and stem cells in different IMAP systems. Their partition was affected by the nature and the concentration of the metal used, but no specificity in distribution for the subpopulations was found.     

Nanoprobe-based immobilized metal affinity chromatography for sensitive and complementary enrichment of multiply phosphorylated peptides

Magnetic nanoparticles (MNP, <100 nm) have rapidly evolved as sensitive affinity probes for phosphopeptide enrichment. By taking advantage of the easy magnetic separation and flexible surface modification of the MNP, we developed a surface‐blocked, nanoprobe‐based immobilized metal ion affinity chromatography (NB‐IMAC) method for the enhanced purification of multiply phosphorylated peptides. The NB‐IMAC method allowed rapid and specific one‐step enrichment by blocking the surface of titanium (IV) ion‐charged nitrilotriacetic acid‐conjugated MNP (Ti⁴⁺‐NTA‐PEG@MNP) with low molecular weight polyethylene glycol. The MNP demonstrated highly sensitive and unbiased extraction of both mono‐ and multiply phosphorylated peptides from diluted β‐casein (2×10^?10 M). Without chemical derivation or fractionation, 1283 phosphopeptides were identified from 400 μg of Raji B cells with 80% purification specificity. We also showed the first systematic comparison on the particle size effect between nano‐sclae IMAC and micro‐scale IMAC. Inductively coupled plasma‐mass spectrometry (ICP‐MS) analysis revealed that MNP had a 4.6‐fold higher capacity for metal ions per unit weight than did the magnetic micro‐sized particle (MMP, 2–10 μm), resulting in the identification of more phosphopeptides as well as a higher percentage of multiply phosphorylated peptides (31%) at the proteome scale. Furthermore, NB‐IMAC complements chromatography‐based IMAC and TiO₂ methods because <13% of mono‐ and 12% of multiply phosphorylated peptide identifications overlapped among the 2700 phosphopeptides identified by the three methods. Notably, the number of multiply phosphorylated peptides was enriched twofold and threefold by NB‐IMAC relative to micro‐scale IMAC and TiO₂, respectively. NB‐IMAC is an innovative material for increasing the identification coverage in phosphoproteomics.     

Single step immobilized metal ion affinity precipitation/chromatography based procedures for purification of concanavalin A and Cajanus cajan mannose-specific lectin

Concanavalin A and a mannose-specific lectin could be precipitated specifically from extracts of jack bean and Cajanus cajan seeds, respectively, using metal charged EGTA. Single step purification of the lectins was also possible using iminodiacetic acid-Sepharose charged with metal ions. Nondenaturing electrophoresis in polyacrylamide gel and that performed in presence of SDS ascertained homogeneity of the isolated lectins. The migration behavior of the purified lectins was comparable with those of the lectins purified using alternative procedures.     

Purification of papain by immobilized metal affinity chromatography (IMAC) on chelating carboxymethyl cellulose

Chelating carboxymethyl cellulose was prepared in bead form by immobilizing iminodiacetic acid on carboxymethyl cellulose which was earlier crosslinked and activated by epichlorohydrin. The prepared matrix was used to purify papain by a factor of 2.6 from commercial papain, and by a factor of 4 from papaya latex by batch adsorption and immobilized metal affinity chromatography respectively. Purification factors obtained were equal in batch mode and double in column mode, to purifications obtained on Chelating Sepharose® Fast Flow. Flow rates up to 38 ml/cm2 h were easily possible on the prepared chelating carboxymethyl cellulose.     

Probing the metal ion selectivity in methionine aminopeptidase via changes in the luminescence properties of the enzyme bound europium ion

We report herein, for the first time, that Europium ion (Eu³⁺) binds to the “apo” form of Escherichia coli methionine aminopeptidase (EcMetAP), and such binding results in the activation of the enzyme as well as enhancement in the luminescence intensity of the metal ion. Due to competitive displacement of the enzyme-bound Eu³⁺ by different metal ions, we could determine the binding affinities of both “activating” and “non-activating” metal ions for the enzyme via fluorescence spectroscopy. The experimental data revealed that among all metal ions, Fe²⁺ exhibited the highest binding affinity for the enzyme, supporting the notion that it serves as the physiological metal ion for the enzyme. However, the enzyme-metal binding data did not adhere to the Irving-William series. On accounting for the binding affinity vis a vis the catalytic efficiency of the enzyme for different metal ions, it appears evident that that the “coordination states” and the relative softness” of metal ions are the major determinants in facilitating the EcMetAP catalyzed reaction.     

Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: separation of synthetic prion peptides

Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of nonporous polymeric beads with the goal of developing a new immobilized metal affinity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin-nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)(2), and (PHGGGWGQ)(4). Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q-->A substitution, or amide nitrogen methylation.     

Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study

Polyvinyldiene fluoride (PVDF)-based affinity membranes with immobilized copper ions were developed in this study. The resulting membranes were tested for their adsorption properties using a model protein, lysozyme, in batch mode. First, different lengths of diamine were utilized as spacer arms to immobilize the metal ions onto the membranes. It was found that the application of 1,8-diaminooctane as the spacer arm led to the highest adsorption capacity. Moreover, the effects of pH and salt concentration were investigated to distinguish the proportion of specific and nonspecific interactions. A big fraction of lysozyme adsorption capacity for the immobilized metal affinity membranes was considered to come from nonspecific electrostatic interactions, which could be reduced by increasing salt concentration. Lastly, the purification of hepatocyte growth factor (HGF) from insect cell supernatant was performed using the immobilized metal affinity membranes in batch mode. HGF was found in the elution condition using EDTA, indicating the successful purification of HGF.     

LdARL-1 His-tagged recombinant protein: purification by immobilized metal affinity expanded bed adsorption

Previously we have cloned three ADP-ribosylation factor-like (ARL) genes from the parasitic protozoan Leishmania donovani: LdARL-3A and 3B, LdARL-1. LdARL-3A was previously purified as an active native form, which was able to bind GTP in vitro. In this paper, we have performed the production and the purification of Histidine-tagged (His-tagged) LdARL-1 recombinant protein by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology. This protein was purified with more than 95% purity and could be successfully used for GTP-binding assay.     

Statistical optimization for immobilized metal affinity purification of secreted human erythropoietin from Drosophila S2 cells

We used a novel approach to affinity purify human erythropoietin (hEPO) following its secretion from Drosophila melanogaster S2 cells. Immobilized metal affinity purification of hEPO was optimized using a two-step serial statistical optimization strategy. After determining the elution conditions (based on preliminary batch-type purification experiments), the first optimization step considered three purification factors; resin, equilibrium, and washing. The results of this analysis showed that the resin amount was the major factor influencing yield and purity in both model equations and the washing factor lowered the confidence limits of the acquired model equations. The washing conditions were then set based on the results of the first step optimization and the second step then optimized three factors; resin, equilibrium, and elution. The yield and purity of hEPO were then compared following purification using three different approaches; batch-type purification based upon the conditions determined by serial statistical optimization, batch-type purification performed in preliminary experiments, and FPLC column chromatography-type purification. We found that the serial statistical optimization approach provided the best combination of yield and purity. These findings indicate that serial statistical optimization strategies can be successfully employed for immobilized metal affinity protein purification using either batch-type or column approaches.