Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.
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PMID:Surface topography of histidine residues in lysozymes. 176 71

Periplasmic expression of recombinant proteins presents many potential benefits that may aid recovery of the protein product. Muramidases are the preferred agents in effecting selective release of recombinant proteins from the periplasm of E. coli and other Gram negative bacteria. Unfortunately cost restricts the use of pure lytic enzymes at large-scale and their removal as process contaminants adds to later purification demands. We constructed a reusable version of bacteriophage T4 lysozyme, by fusing a His-Gln-(His)3 peptide sequence to the C-terminus of a cysteine-free pseudo wild type bacteriophage T4 lysozyme. The peptide tail allowed rapid and high-level recovery on IDA Sepharose columns charged with Zn2+, Ni2+ and Cu2+ ions. The binding to metal-charged supports was specifically mediated by the histidine-rich tail as no binding was observed for the original cysteine-free pseudo wild type lysozyme. The strength of retention of polyhistidine recombinant T4 lysozyme on charged supports followed the expected Cu > Ni > Zn pattern, but there were few differences in the levels of purity and recovery of the modified enzyme, from columns charged with the different metal ions.
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PMID:Expression and purification of a recombinant metal-binding T4 lysozyme fusion protein. 887 73

An investigation into the adsorption behavior under batch equilibrium binding conditions of hen egg white lysozyme (HEWL) with the immobilized metal ion affinity chromatographic adsorbent, Cu(2+)-IDA Sepharose CL-4B, in the presence of high concentrations of NaCl or KCl has been undertaken. When the concentration of NaCl or KCl in the adsorption buffer was < or = 0.2 M, the adsorption data correlated with predictions based on the Langmuir model. However, when the salt concentration was > or = 0.5 M, the adsorption data appeared to follow the Freundlich-Langmuir model under these higher ionic strength conditions. Under elevated ionic strength conditions, the protein-ligand interactions were shown to involve positive cooperativity by Scatchard plot analysis of the experimental data. The number, n, of interacting protein molecules increased with the salt concentration, while the apparent dissociation constant Kd under these high ionic strength conditions was less dependent on the type of salt present in the adsorption buffer. These studies reveal that the binding of Cu(2+)-IDA Sepharose CL-4B to HEWL acceptor molecules under conditions of high ionic strength is characteristic of general heterogeneous interactions, whereby the immobilized metal ion affinity chromatography (IMAC) ligands bind to a protein acceptor undergoing isodesmic indefinite self-association and multisite attachment. As a consequence, these results on the adsorption behavior of HEWL have important general implications for the preparative use of immobilized metal ion affinity adsorbents in packed and expanded bed systems, particularly with regard to the resolution and productivity of the separation for the target protein. In addition, the results from these studies are relevant to investigations involving analytical affinity chromatographic or biosensor assessment of the interaction of proteins with immobilized IMAC ligands when high ionic strength equilibration feedstocks or conditions are employed.
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PMID:Protein interaction with immobilized metal ion affinity ligands under high ionic strength conditions. 892 63

The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents. Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E. coli extracts in a single step. Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose. The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale. By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.
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PMID:Characterisation of non-porous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E. coli extract. 918

In this investigation, employing a highly sensitive microcalorimeter, we measure the influence of pH value and salt concentration on the heat of interaction between lysozyme and CS-IDA-Cu(II) gel. The direct enthalpy measurement of the interaction provides thermodynamic information regarding the binding behavior of lysozyme toward the immobilized metal ion. The binding enthalpy altered by adsorbed lysozyme at various pH values and salt concentrations are measured. The findings, along with the reported binding isotherm, are discussed herein.
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PMID:Microcalorimetric Studies of the Interactions of Lysozyme with Immobilized Cu(II): Effects of pH Value and Salt Concentration 924 Nov 40

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the purification of natural and recombinant therapeutic products and is beginning to find industrial applications. The design, optimization, and scale-up of a chromatographic process using IMAC demands a thorough understanding to be developed regarding the fundamental factors governing the various interactions between immobilized metal ions and proteins. Consequently, there is an immediate need to find out a theory that is able to account for these interactions most efficiently in a qualitative as well as a quantitative manner. In view of this requirement, the interactions of several model proteins (lysozyme, ovalbumin, bovine serum albumin, conalbumin, and wheat germ agglutinin) with metal (Cu(II), Ni(II))-chelated IDA (iminodiacetate) and tris(2-aminoethyl)amine were investigated. The adsorption data were analyzed using four isotherm models, viz., the general affinity interaction theory/Langmuir model, the Freundlich model, the Temkin model, and the Langmuir-Freundlich model, and the sorption parameters were computed. Although the first three models were applicable to some protein-IMA-M(II) systems, the Langmuir-Freundlich model appeared to be the most efficient model for explaining the interactions of proteins with IMA-M(II) gels. Also, this model was able to explain cooperativity and binding heterogeneity in quantitative terms. It is envisaged that this analysis would be useful in developing an improved understanding of protein-immobilized metal ion interactions and providing guidelines for designing preparative-scale separations using IMAC.
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PMID:Interactions of proteins with immobilized metal ions: a comparative analysis using various isotherm models. 1115 83

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).
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PMID:Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography. 1121 18

A new approach, combining metal coordination with the molecular imprinting technique, was developed to prepare affinity materials. Magnetic poly(glycidyl methacrylate) microspheres in monosize form were used for specific recognition toward the target protein. The magnetic poly(glycidyl methacrylate) microspheres were prepared by dispersion polymerization in the presence of magnetite nanopowder. Surface imprinted magnetic poly(glycidyl methacrylate) microspheres based on metal coordination were prepared and used for the selective recognition of human serum albumin. Iminodiacetic acid was used as the metal coordinating agent and human serum albumin was anchored by Cu(2+) ions on the surface of magnetic poly(glycidyl methacrylate) microspheres by metal coordination. The magnetic poly(glycidyl methacrylate) microspheres were coated with a polymer formed by condensation of tetraethyl orthosilicate and 3-aminopropyltrimethoxysilane. The human serum albumin imprinted magnetic poly(glycidyl methacrylate) microspheres were characterized by scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy and particle size analysis. The maximum adsorption capacity of human serum albumin imprinted magnetic poly(glycidyl methacrylate) microspheres was 37.7 mg/g polymer at pH 6.0. The selectivity experiments of human serum albumin imprinted magnetic poly(glycidyl methacrylate) microspheres prepared with different concentrations in the presence of lysozyme, bovine serum albumin and cytochrome C were performed in order to determine the relative selectivity coefficients.
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PMID:Surface molecularly imprinted magnetic microspheres for the recognition of albumin. 2482 45

In this study, we reported iminodiacetic acid-copper ion complex (IDA-Cu) immobilized onto gold nanoparticles (GNPs)-modified glassy carbon electrode as a novel electrochemical platform for selective and sensitive determination of lysozyme (Lys). IDA-Cu complex acted as an efficient recognition element capable of capturing Lys molecules. GNPs acts as a substrate to immobilize IDA-Cu coordinative complex and its interaction with Lys leds to a great signal amplification through measuring changes in differential pulse voltammetric (DPV) peak current of [Fe(CN)6](3-/4-) redox probe. Upon the recognition of the Lys to the IDA-Cu, the peak current decreased due to the hindered electron transfer reaction on the electrode surface. Under optimum condition, it was found that the proposed method could detect Lys at wide linear concentration range (0.1 pM to 0.10 mM) with detection limit of 60 fM. Furthermore, electrochemical impedance spectroscopy (EIS) detection of Lys was demonstrated as a simple and rapid alternative analytical technique with detection limit of 80 fM at concentration range up to 0.1mM. In addition, the proposed sensor was satisfactorily applied to the determination of Lys in real samples such as hen egg white. The proposed modified electrode showing the high selectivity, good sensitivity and stability toward Lys detection may hold a great promise in developing other electrochemical sensors based on metal-chelate affinity complexes.
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PMID:Novel voltammetric and impedimetric sensor for femtomolar determination of lysozyme based on metal-chelate affinity immobilized onto gold nanoparticles. 2614 67

The Cu2+-decorated functional mesoporous material was fabricated by thermally initiated free-radical polymerization of octavinyl polyhedral oligomeric silsesquioxane. It was used as adsorbent for highly specific separation of histidine (His)-rich proteins in blood and cell lysate based on immobilized metal affinity chromatography. The functional mesoporous material (named as PPOSS-IDA-Cu2+) was characterized in detail and its selectivity and binding capacity were evaluated using a His-rich protein (bovine hemoglobin, BHb) and other proteins (bovine serum albumin, myoglobin, lysozyme and horseradish peroxidase) containing fewer surface-exposed His residues as model proteins. The results indicated that PPOSS-IDA-Cu2+ exhibited large specific surface area and good selective adsorption ability and the maximum adsorption capacity for BHb was 3150mgg-1. Moreover, PPOSS-IDA-Cu2+ had excellent recyclability and the adsorption capacity of the reused material for BHb remained almost unchanged after six cycles. In addition, PPOSS-IDA-Cu2+ not only showed excellent performance for the removal of highly abundant hemoglobin in human blood, but also can be a good adsorbent for the enrichment of proteins in cell lysate. It was the first time to explore the application of Cu2+-decorated functional material as an adsorbant for the separation of proteins in cell lysate. This approach can be combined with other techniques which can remove or deplete highly abundant proteins from real biological samples to obtain more comprehensive data about low abundant His-rich proteins in proteomic analysis.
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PMID:Facile synthesis of copper(II)-decorated functional mesoporous material for specific adsorption of histidine-rich proteins. 2891 56


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