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Metal Ion Affinity Chromatography (metal + ion_affinity_chromatography)

Distribution by Scientific Domains

Chemistry	71%

Kinds of Metal Ion Affinity Chromatography

immobilized metal ion affinity chromatography

Selected Abstracts

Separation of Pure and Immunoreactive Virus-Like Particles Using Gel Filtration Chromatography Following Immobilized Metal Ion Affinity Chromatography

BIOTECHNOLOGY PROGRESS, Issue 2 2001
Yu-Shen Cheng
A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni2+) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm3. However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus. [source]

A Generic Approach to Monofunctionalized Protein-Like Gold Nanoparticles Based on Immobilized Metal Ion Affinity Chromatography

CHEMBIOCHEM, Issue 4 2006
Raphaël Lévy Dr.
Control of a peptide-capped gold-nanoparticle (NP) surface with single-molecule accuracy is demonstrated. Immobilized metal ion affinity chromatography (IMAC) has been used to separate peptide-capped NPs as a function of the number of molecular labels (see scheme). The method described in this paper is simple, quantitative and directly applicable to the preparation of monofunctionalized nanoparticles with any water-soluble chemical moieties. [source]

Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulis

FEBS JOURNAL, Issue 16 2000
Bingze Xu
A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source]

Oligohis-tags: mechanisms of binding to Ni2+ -NTA surfaces

JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2009
Steven Knecht
Abstract Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni2+ immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C - or N -terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni2+ -NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni2+ -NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (KD) of 14,±,1,nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2005
Kris Gevaert Professor
Abstract We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+ -immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O ,18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190,phosphorylated peptides from 152,different proteins. This dataset includes 38,novel protein phosphorylation sites. [source]

A Generic Approach to Monofunctionalized Protein-Like Gold Nanoparticles Based on Immobilized Metal Ion Affinity Chromatography

Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5

CLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2006
Å. Marknell DeWitt
Summary Background Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein. Objective To clone and characterize timothy grass pollen allergen Phl p 4. Methods Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3,-and 5,-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies. Results Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner. Conclusions Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4. [source]