sepharose and iminodiacetic-acid

This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.

Topics: Adsorption; Chelating Agents; Chromatography, Affinity; Coordination Complexes; Copper; Gels; Green Fluorescent Proteins; Histidine; Imino Acids; Metals; Nickel; Oligopeptides; Protein Refolding; Proteins; Recombinant Proteins; Resins, Synthetic; Sepharose

Metal ion affinity chromatography is widely used to purify peptides on the basis of the dissimilarities of their amino acids. However, researchers are interested in the separation differences between different metal ions in this method. In our study, four kinds of commonly used metal ions are compared by the amount of immobilized metal ion on iminodiacetic acid-Sepharose and binding amount of soybean peptide to immobilized iminodiacetic acid-Mn(+) adsorbents and evaluated by high-performance liquid chromatography (HPLC) profiles. The results show that due to the different adsorption behaviors of metal ions, the binding ability order of soybean protein peptide on the column should be Fe(3+) > Cu(2+) > Zn(2+) > Ca(2+). The HPLC profiles show that peptides adsorbed by four kinds of metal ions display similar strong hydrophobic characteristics.

Topics: Adsorption; Analysis of Variance; Chromatography, Affinity; Chromatography, High Pressure Liquid; Hydrophobic and Hydrophilic Interactions; Imino Acids; Metals, Heavy; Peptides; Protein Binding; Sepharose; Soybean Proteins

This paper describes some recent advances in the methodology of immobilized metal ion affinity gel electrophoresis. Four different ways to incorporate metal chelate ligands in agarose and polyacrylamide-based electrophoresis gels are evaluated, a new polymerizable metal chelating ligand, allyl-2-hydroxy-3-(N,N-dicarboxymethyl)amino-propyl ether, is introduced, and the determination of affinity constants described. The affinities of model proteins (ribonucleases A and B and cytochromes c from different species) for the transition metal chelate iminodiacetic acid-Cu(II) were studied. The results were found to be in agreement with literature data on immobilized metal ion affinity chromatography, and the polymer nature and the different chemistries used influenced the affinity only quantitatively, keeping the basic mechanisms of interaction unchanged.

Topics: Animals; Candida; Cattle; Chelating Agents; Copper; Cytochrome c Group; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Imino Acids; Metals; Molecular Structure; Ribonuclease, Pancreatic; Ribonucleases; Sepharose; Tuna

Topics: Amino Acid Sequence; Apoproteins; Chelating Agents; Chromatography, Affinity; Copper; Hydrogen-Ion Concentration; Imino Acids; Lactoferrin; Metals; Molecular Sequence Data; Molecular Structure; Nickel; Peptides; Proteins; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Zinc

The Zn(II)-binding site from the active center of human carbonic anhydrase II, formed by three His side chains, can be grafted onto the recombinant serum retinol-binding protein (RBP). The artificial binding site in the resulting variant RBP/H3(A) has high affinity for Zn(II) and stabilizes the protein against denaturation.. The metal-ion specificity of the grafted Zn(II) binding site in RBP/H3(A) was investigated. Both Cu(II) and Ni(II) bound with high affinity, although the Kd values were not as low as for Zn(II) binding. Competition experiments with the chelate ligands iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) suggested that both Ni(II) and Cu(II) bound to the protein in an octahedral manner with three vacant coordination sites, as previously observed for Zn(II). A substituted pyrrolidine-dicarboxylic acid was designed as a structurally rigid IDA compound and coupled to a matrix. Using this support in an immobilized metal affinity chromatography (IMAC), RBP/H3(A) was purified from the bacterial cell extract in one step with unprecedented efficiency.. Although the His3 metal-binding site used here had been removed from the substrate pocket of an enzyme and exposed to solvent on a protein surface, it showed clear selectivity for Zn(II) compared to Cu(II) and Ni(II). Thus the properties of this structurally defined metal-binding site (which are not shared by isolated His residues or flexible oligo-His tags) can be preserved when it is added to proteins. An IMAC matrix with improved behaviour was designed, allowing highly selective purification of RBP/H3(A) and of His6-tagged RBP as well. Such rational design of supramolecular recognition may be generally useful in the fields of protein engineering and drug design.

Topics: Binding Sites; Carbonic Anhydrases; Chromatography, Affinity; Escherichia coli; Histidine; Humans; Imino Acids; In Vitro Techniques; Ligands; Models, Molecular; Molecular Structure; Protein Conformation; Protein Engineering; Recombinant Fusion Proteins; Retinol-Binding Proteins; Sepharose; Zinc

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 range pH 5.5-8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized Mn+-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-metal-ion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surface-exposed aspartic and glutamic acid residues on the protein with the immobilized Mn+-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 of clustering of the surface-accessible aspartic and glutamic 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.

Topics: Animals; Aspartic Acid; Buffers; Chickens; Chromatography, Affinity; Cytochrome c Group; Dogs; Female; Glutamic Acid; Horses; Hydrogen-Ion Concentration; Imino Acids; Metals; Muramidase; Myoglobin; Proteins; Sepharose; Sheep; Species Specificity; Surface Properties

Topics: Biotechnology; Chelating Agents; Chromatography, Affinity; Imino Acids; Metals; Proteins; Sepharose

Conditions for the immobilization of O-phosphoserine (OPS) to epoxy-activated Sepharose CL-4B are described. The binding behaviour of OPS and iminodiacetic acid (IDA) immobilized onto Sepharose CL-4B, toward the hard Lewis metal ions Al3+, Fe3+, Ca2+ and Yb3+, and Cu2+ ion as a borderline metal ion control, over the pH range pH 4.0 to pH 8.0, was examined. Immobilized OPS shows a stronger affinity for Fe3+ and Al3+ ions but a lower affinity for Cu2+ and Yb3+ ions, compared to immobilized iminodiacetic acid (IDA), over the equilibrating range examined. Immobilized OPS-Mn+ was screened for protein binding using as model proteins tuna heart cytochrome c (THCC), horse myoglobin (HMYO) and hen egg while lysozyme (HEWL) over the pH range 5.5 to 8.0. Immobilized OPS-Fe3+ bound THCC under all the examined equilibrating conditions, bound HMYO between pH 5.5 and pH 7.0 and did not bind HEWL under any condition examined. Immobilized OPS thus presents an additional mode of metal ion and protein selectivity in immobilized-metal affinity chromatography.

Topics: Amino Acids; Animals; Chelating Agents; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cytochrome c Group; Egg White; Horses; Imino Acids; Ligands; Metals; Myocardium; Phosphoserine; Sepharose; Temperature; Tuna

The metal-binding properties of partially purified untransformed or salt-dissociated bovine estrogen receptors were studied using zinc-chelated iminodiacetic acid gels. Only the salt-dissociated 5S receptor is retained by the metal-chelated resin, and this interaction is dependent on the presence of dithiothreitol. The untransformed 9S receptor is not retained, indicating that the zinc-interacting amino acid residues may be masked by receptor-associated proteins such as 90K heat-shock protein or because of an unfavorable receptor conformation.

Topics: Animals; Cattle; Chelating Agents; Chromatography, Affinity; Cytosol; Dithiothreitol; Gels; Heparin; Imino Acids; Receptors, Estrogen; Scintillation Counting; Sepharose; Ultracentrifugation; Zinc

The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.

Topics: Blood Protein Electrophoresis; Blood Proteins; Buffers; Chromatography, Affinity; Cobalt; Copper; Electrophoresis, Gel, Two-Dimensional; Humans; Hydrogen-Ion Concentration; Imino Acids; Isoelectric Focusing; Manganese; Metals; Nickel; Osmolar Concentration; Salts; Sepharose; Zinc

A method is described to purify pancreatic carboxypeptidases B (CPB), removing contaminating endoproteinases that interfere with use of CPB for carboxy-terminal analysis or modification of proteins. The separation uses zinc chelate chromatography and is based on the property that CPB has higher affinity for immobilized zinc ions than do serine proteinases such as trypsin and chymotrypsin, which are abundant endoproteolytic activities in pancreas. CPB preparations are loaded onto a column of iminodiacetic acid-Sepharose that has been saturated with ZnCl2. A step gradient with buffers of decreasing pH is used to elute bound proteins. CPB elutes at a lower pH than do the serine proteinases.

Topics: Buffers; Carboxypeptidases; Chelating Agents; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Hydrogen-Ion Concentration; Imino Acids; Sepharose; Zinc