Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.
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PMID:Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces. 1685 44

Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 angstroms using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2(1) (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 angstroms, beta = 102.97 degrees) and the orthorhombic space group P2(1)2(1)2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 angstroms), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.
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PMID:Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica. 1688 May 47

A blend mixture of biodegradable poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol)-NH(2) (PLGA-b-PEG-NH(2)) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.
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PMID:Surface functionalized electrospun biodegradable nanofibers for immobilization of bioactive molecules. 1688 87

Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5x enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35x higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na(2)SO(4) as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9x (germ) and 5x (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.
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PMID:Aqueous two-phase extraction for protein recovery from corn extracts. 1692 Apr 13

The performance of molecularly imprinted polymers (MIPs) is of interest to researchers in the field of analytical chemistry, and in the pharmaceutical and food industries. Because the choice of the functional monomer(s) plays a key role in the selectivity of a MIP, the synthesis of an effective, tight-binding MIP can be difficult and time-consuming, involving the evaluation of the binding performance of MIPs of many different compositions. In this study, we report an express method combining molecular imprinting and microcontact printing techniques to prepare a polymer thin film as an artificial antibody. In addition to the microcontact printing technique, isothermal titration of monomers to proteins stamps was investigated to screen the functional monomer for MIPs. Finally, the importance of the choice of cross-linking monomers in MIPs was studied, and these studies suggest that monomers containing an optimal length PEG spacer give higher imprinting effectiveness. Several model antigens (lysozyme, ribonuclease A and myoglobin) were adsorbed on a cover glasses that were pretreated with hexamethyldisilazane (HMDS). These protein stamps were then contacted with different monomer solutions (cross-linking monomers) on a glass slide substrate. Photopolymerization yielded the molecularly imprinted polymer. This technique, analogous to microcontact printing, allows for the rapid, parallel synthesis of MIPs of different compositions, and requires very small volumes of monomers (ca. 4 microL). The technique also avoids potential solubility problems with the molecular targets. Of several cross-linking monomers screened, tetraethyleneglycol dimethacrylate (TEGDMA) gave the most selective lysozyme binding, while polyethyleneglycol 400 dimethacrylate (PEG400DMA) were most selective for ribonuclease A and myoglobin.
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PMID:The microcontact imprinting of proteins: the effect of cross-linking monomers for lysozyme, ribonuclease A and myoglobin. 1697 44

Surface-induced aggregation is a common instability during protein storage, delivery and purification. This aggregation can lead to the formation of fibrils rich in intermolecular beta-sheet structure. Techniques to probe surface-clustering are limited. Here we use protein intrinsic fluorescence and thioflavin T probe fluorescence in a total internal reflection fluorescence (TIRF) sampling geometry to simultaneously monitor the kinetics of adsorption and aggregation for chicken egg lysozyme on a silica surface. We observe a slow surface-induced aggregation process that continues well after the lysozyme adsorption kinetics have plateaued. The rate of surface-induced aggregation is independent of the lysozyme concentration in solution. Consistent with the clustering observed via thioflavin T fluorescence, infrared amide I band spectra also show a 1.5-fold increase in intermolecular beta-sheet content upon lysozyme adsorption. Tryptophan emission spectra show no evidence for any tertiary structural change upon adsorption. Furthermore, we observe that the covalent modification of lysozyme with a single poly(ethylene glycol) (PEG) grafted chain does not inhibit aggregation on the surface, but a second PEG graft significantly inhibits the intermolecular beta-sheet formation.
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PMID:Aggregation of lysozyme and of poly(ethylene glycol)-modified lysozyme after adsorption to silica. 1731 16

Herein we report a new strategy for protein refolding by taking advantage of the unique surface and pore characteristics of ethylene-bridged periodic mesoporous organosilica (PMO), which can effectively entrap unfolded proteins and assist refolding by controlled release into the refolding buffer. Hen egg white lysozyme was used as a model protein to demonstrate the new method of protein refolding. Through loading of denatured proteins inside uniform mesoporous channels tailored to accommodate individual protein, protein aggregation was minimized, and the folding rate was increased. Poly(ethyleneglycol) (PEG)-triggered continuous release of entrapped denatured lysozyme allowed high-yield refolding with high cumulative protein concentrations. The new method enhances the oxidative refolding of lysozyme (e.g., over 80% refolding yield at about 0.6 mg/mL).
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PMID:Protein refolding assisted by periodic mesoporous organosilicas. 1740 59

It was the aim of this study to establish triglyceride matrices as potential carriers for long-term release of brain-derived neurotrophic factor (BDNF), a potential therapeutic for Huntington's disease. First, four different manufacturing strategies were investigated with lysozyme as a model substance: either lyophilized protein was mixed with lipid powder, or suspended in organic solution thereof (s/o). Or else, an aqueous protein solution was dispersed by w/o emulsion in organic lipid solution. Alternatively, a PEG co-lyophilization was performed prior to dispersing solid protein microparticles in organic lipid solution. After removal of the solvent(s), the resulting powder formulations were compressed at 250 N to form mini-cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight. Protein integrity after formulation and release was evaluated from an enzyme activity assay and SDS-PAGE. Confocal microscopy revealed that the resulting distribution of FITC-lysozyme within the matrices depended strongly on the manufacturing method, which had an important impact on matrix performance: matrices with a very fine and homogeneous protein distribution (PEG co-lyophilization) continually released protein for 2 months. The other methods did not guarantee a homogeneous distribution and either failed in sustaining release for more than 1 week (powder mixture), completely liberating the loading (s/o dispersion) or preserving protein activity during manufacturing (w/o emulsion, formation of aggregates and 25% activity loss). Based on these results, miniature-sized implants of 1 mm diameter, 0.8 mm height and 1 mg weight were successfully loaded by the PEG co-lyophilization method with 2% BDNF and 2% PEG. Release studies in phosphate buffer pH 7.4 at 4 and 37 degrees C revealed a controlled release of either 20 or 60% intact protein over one month as determined by ELISA. SDS-PAGE detected only minor aggregates in the matrix during release at higher temperature. In vivo evaluation of lipid cylinders in the striatum of rat brains revealed a biocompatibility comparable to silicone reference cylinders.
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PMID:Towards controlled release of BDNF--manufacturing strategies for protein-loaded lipid implants and biocompatibility evaluation in the brain. 1742 70

Previous studies have shown that stereocomplexed hydrogels are rapidly formed in situ by mixing aqueous solutions of eight-arm poly(ethylene glycol)-poly(L-lactide) and poly(ethylene glycol)-poly(D-lactide) star block copolymers (denoted as PEG-(PLLA)(8) and PEG-(PDLA)(8), respectively). In this study, in vitro and in vivo protein release from stereocomplexed hydrogels was investigated. These hydrogels were fully degradable under physiological conditions. Proteins could be easily loaded into the stereocomplexed hydrogels by mixing protein containing aqueous solutions of PEG-(PLLA)(8) and PEG-(PDLA)(8) copolymers. The release of the relatively small protein lysozyme (d(h)=4.1 nm) followed first order kinetics and approximately 90% was released in 10 days. Bacteria lysis experiments showed that the released lysozyme had retained its activity. The relatively large protein IgG (d(h)=10.7 nm) could be released from stereocomplexed hydrogels with nearly zero order kinetics, wherein up to 50% was released in 16 days. The in vitro release of the therapeutic protein rhIL-2 from stereocomplexed hydrogels also showed nearly zero order kinetics, wherein up to 45% was released in 7 days. The therapeutic efficacy of stereocomplexed hydrogels loaded with 1x10(6) IU of rhIL-2 was studied using SL2-lymphoma bearing DBA/2 mice. The PEG-(PLLA)(8)/PEG-(PDLA)(8)/rhIL-2 mixture could be easily injected intratumorally. The released rhIL-2 was therapeutically effective as the tumor size was reduced and the cure rate was 30%, whereas no therapeutic effect was achieved when no rhIL-2 was given. However, the cure rate of rhIL-2 loaded stereocomplexed hydrogels was lower, though not statistically significant, compared to that of a single injection with 1x10(6) IU of free rhIL-2 at the start of the therapy (cure rate=70%). The therapeutic effect of rhIL-2 loaded stereocomplexed hydrogels was retarded for approximately 1-2 weeks compared to free rhIL-2, most likely due to a slow, constant release of rhIL-2 from the hydrogels.
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PMID:In vitro and in vivo protein delivery from in situ forming poly(ethylene glycol)-poly(lactide) hydrogels. 1747 60

Cyclodextrin-PEG hydrogels were prepared by reaction of hexamethlyene isocyanate-activated beta-cyclodextrins with 1.9kDa NH(2)PEGNH(2). The reaction was carried out in anhydrous dimethylsulfoxide by using 0.25:1, 0.33:1, 0.5:1, 0.67:1, 1:1, and 2:1 CD/PEG molar ratios. The addition of acetic acid to the reaction mixture was found to slow the cross-linking reaction, yielding homogeneous matrices. The mechanical characterization indicated that the elasticity of the matrices increased as the CD content in the hydrogel increased while the elongation was irrespective of the hydrogel composition. By incubation in water and ethanol, the hydrogels underwent complete swelling in 5-10min. The water up-take increased logarithmically as the CD/PEG ratio decreased to reach a swelling degree of 800% (swollen hydrogel/dry hydrogel, w/w%). The ethanol uptake increased with a power correlation as the CD/PEG ratio decreased to reach a swelling degree of about 1000% with 0.25:1 CD/PEG hydrogel. Lysozyme, beta-estradiol, and quinine were loaded by swell embedding. The lysozyme loading increased as the CD/PEG ratio decreased while the incorporation of beta-estradiol and quinine displayed inverse correlation with respect to the CD/PEG ratio. The maximal incorporation (loaded drug/dry hydrogel, w/w%) for lysozyme, beta-estradiol and quinine was 2, 0.6, and 2.4%, respectively. Lysozyme was quickly released from the matrices, and the release was faster as the CD/PEG ratio decreased. Also, beta-estradiol and quinine release rates were inversely proportional to the CD/PEG ratio, but in these cases, the release profiles were strongly affected by the drug interaction with the hexamethylated beta-cyclodextrins in the matrices.
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PMID:Cyclodextrin/PEG based hydrogels for multi-drug delivery. 1759 13


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