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)

New PEG derivatives were studied for peptide and protein modification, based upon an amino acid arm, Met-Nle or Met-beta Ala, activated as succinimidyl ester. PEG-Met-Nle-OSu or PEG-Met-beta Ala-OSu react with amino groups in protein-yielding conjugates with stable amide bond. From these conjugates PEG may be removed by BrCN treatment, leaving Nle or beta Ala as reporter amino acid, at the site where PEG was bound. The conjugation of PEG and its removal by BrCN treatment was assessed on a partial sequence of glucagone and on lysozyme as model peptide or protein. Furthermore, insulin, a protein with three potential sites of PEGylation, was modified by PEG-Met-Nle, and the PEG isomers were separated by HPLC. After removal of PEG, as reported above, the sites of PEGylation were identified by characterization of the two insulin chains obtained after reduction and carboxymethylation. Mass spectrometry, amino acid analysis and Edman sequence, could reveal the position of the reporter norleucine that corresponds to the position of PEG binding.
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PMID:New PEGs for peptide and protein modification, suitable for identification of the PEGylation site. 1117 Mar 67

Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the epsilon-amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.
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PMID:Drug delivery systems employing 1,6-elimination: releasable poly(ethylene glycol) conjugates of proteins. 1131 76

The effect of potassium thiocyanate on the partitioning of lysozyme and BSA in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system has been investigated. As a result of the addition of potassium thiocyanate to the PEG/ammonium sulfate system, the PEG/mixed salts aqueous two-phase system was formed. It was found that the potassium thiocyanate could alter the pH difference between the two phases, and, thus, influence the partition coefficients of the differently charged proteins. The relationship between partition coefficient of the proteins and pH difference between two phases has been discussed. It was proposed that the pH difference between two phases could be employed as the measurement of electrostatic driving force for the partitioning of charged proteins in polyethylene 2000/ammonium sulfate aqueous two-phase system.
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PMID:The effect of KSCN on the partition of proteins in polyethylene glycol/(NH4)2SO4 aqueous two-phase system. 1132 Nov 61

Compartmentalization of enzymes in the nanometric-scaled container, to improve their stability and availability, has recently attracted a strong interest in the field of pharmaceutics. In this study, the enzymatic activity of lysozyme in the core of polyion complex (PIC) micelles, which were formed from egg white lysozyme and poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copolymer (PEG-P(Asp)), was evaluated using a colorimetric method. Apparent enzymatic activity of lysozyme entrapped in the core of PIC micelles remarkably increased compared to that of free lysozyme, which is mainly attributed to a decrease in the observed Michaelis constant (K(m,obs)). The reciprocal of the K(m,obs) values nicely correlated to the corona thickness of PIC micelles, suggesting that the corona layer of PIC micelle may act as the reservoir of the substrate, p-nitrophenyl penta-N-acetyl-beta-chitopentaoside. This result indicates that the enzymatic activity can be controlled by changing the corona thickness of PIC micelles through a variation in the mixing ratio of PEG-P(Asp) to lysozyme. This type of PIC micelle system entrapping enzyme in the core might be useful for the design of diagnostic as well as targetable therapeutic systems of enzyme including antibody-directed enzyme prodrug therapy (ADEPT).
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PMID:Pronounced activity of enzymes through the incorporation into the core of polyion complex micelles made from charged block copolymers. 1138 87

Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate protein carriers in order to overcome mucosal barriers. We have attempted to combine both issues through the conception of new biodegradable polymer nanoparticles: (i) poly(ethylene glycol) (PEG)-coated poly(lactic acid) (PLA) nanoparticles, chitosan (CS)-coated poly(lactic acid-glycolic acid (PLGA) nanoparticles and chitosan (CS) nanoparticles. These nanoparticles have been tested for their ability to load proteins, to deliver them in an active form, and to transport them across the nasal and intestinal mucosae. Additionally, the stability of some of these nanoparticles in simulated physiological fluids has been studied. Results showed that the PEG coating improves the stability of PLA nanoparticles in the gastrointestinal fluids and helps the transport of the encapsulated protein, tetanus toxoid, across the intestinal and nasal mucosae. Furthermore, intranasal administration of these nanoparticles provided high and long-lasting immune responses. On the other hand, the coating of PLGA nanoparticles with the mucoadhesive polymer CS improved the stability of the particles in the presence of lysozyme and enhanced the nasal transport of the encapsulated tetanus toxoid. Finally, nanoparticles made solely of CS were also stable upon incubation with lysozyme. Moreover, these particles were very efficient in improving the nasal absorption of insulin as well as the local and systemic immune responses to tetanus toxoid, following intranasal administration. In summary, these results show that a rational modification in the composition and structure of the nanoparticles, using safe materials, increases the prospects of their usefulness for mucosal protein delivery and transport.
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PMID:Design of biodegradable particles for protein delivery. 1177 45

The objective of this study was designed to extend the application of poly(epsilon-caprolactone) (PCL) in delivery of macromolecular proteins. The strategy applied here is to create a porous structure in PCL films in order to control the diffusion rate of protein. Various amounts of both high-molecular-weight and low-molecular-weight poly(ethylene glycol) (PEG) were used as pore-forming agents. The porous films were prepared by a solvent-casting-leaching method. The thicknesses of the prepared films were controlled to be in the range of 75.3 +/- 0.6 similar 81.7 +/- 0.6 mum. The pore fraction of films was determined to be 27.7 +/- 1.0% similar 52.5 +/- 0.8% for PEG(10000) and 26.6 +/- 1.8% similar 48.8 +/- 1.4% for PEG(4000). The pore fraction initially increased with increasing amounts of PEG, independent of the molecular weight of PEG. In the permeation study, lysozyme was used as a model diffuser. The permeation rate of protein increased as the pore fraction of films increased, especially when 30 similar 40% of PEG was added initially, and this phenomenon was more prominent when low-molecular-weight PEG was used. This result was probably due to the highly porous structure creating interconnected channels in the films, further enhancing protein diffusion. In addition, the size of micropores formed by PEG(4000) was observed to be larger than by PEG(10000), which also accounted for faster permeation rate of lysozyme through PCL-PEG(4000) porous films.
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PMID:Permeation of protein from porous poly(epsilon-caprolactone) films. 1187 Jun 57

Diffusion has a central role in protein crystal growth both in microgravity conditions and on ground. Recently several reports have been focused on the importance to use the generalized Fick's equations in n-component systems where crystals grow. In these equations the total flux of each component is produced by the own concentration gradient (main flow) and by the concentration gradient of the other components (cross-flow) present in the system. However in literature the latter effect is often neglected, and the so-called pseudo-binary approximation is used. Lin et al. (1995) proposed a mathematical model to evaluate the concentration profile of the species present around a growing protein crystal. Although the model is reliable, it suffers of the pseudo-binary approximation (neglecting cross term diffusion coefficients and using binary diffusion coefficients), probably because of the lack of multicomponent diffusion data. The present model is based on the experimental set-up proposed by Lin et al. (1995). Nevertheless we have included the coupled diffusion effects, according to the correct description of the matter transport through the generalized Fick's equations. The crystal growth rate is calculated for different gravity levels. The model has been applied to the ternary lysozyme-NaCl-water and quaternary lysozyme-poly(ethylene glycol) (PEG)-NaCl-water systems using recent diffusion data.
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PMID:Analysis of the influence of coupled diffusion on transport in protein crystal growth for different gravity levels. 1235 76

The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG coatings were prepared by grafting methoxy-terminated aldehyde-PEG (MW 5000 Da) onto two amino-functionalised surfaces with different amine group densities, generated by radio frequency glow discharge polymerisation of n-heptylamine and allylamine. Grafting was performed at the lower critical solution temperature to maximise the graft density of the PEG chains. XPS showed that the grafted density of PEG chains was slightly higher on the allylamine surface. XPS detected no adsorption of either protein on either PEG coating. ToF-SIMS analysis, on the other hand, found, in the positive ion spectra, minute but statistically significant signals assignable to amino acid fragment ions from both proteins adsorbed to the lower density PEG coating and from LYS but not FN on the higher density PEG coating. Negative ion spectra contained relatively more intense protein fragment ion signals for the lower density PEG coating but no changes assignable to adsorbed proteins on the higher density PEG coating. These results demonstrate the importance of utilising highly sensitive techniques to study protein adsorption on surfaces intended to be protein resistant, and that both positive and negative ion ToF-SIMS spectra should be acquired to probe for possible very low levels of protein adsorption.
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PMID:Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry. 1236 16

The identification of PEGylation sites is essential in the characterization of PEGylated therapeutic proteins. This report describes a simple and novel method of finding poly(ethylene glycol) (PEG) conjugation sites in PEGylated proteins by using a hetero-functional biotin-PEG-N-hydroxyl succinimide derivative. PEGylated lysozyme species having a biotin moiety at each PEG chain end were separated and digested by trypsin. Among the digested lysozyme fragments, biotin-terminated PEGylated peptide fragments were purified by a monomeric avidin immobilized column. Their mass was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry, directly indicating that PEG was conjugated to lysine 33, 97, 116 residues. Reversed-phase high-pressure liquid chromatography results for the PEGylated peptide fragments exhibited that PEGylation occurred preferentially at lysine 33> lysine 97> lysine 116.
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PMID:A novel method for identifying PEGylation sites of protein using biotinylated PEG derivatives. 1248 86

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.
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PMID:Tobacco protein separation by aqueous two-phase extraction. 1264 Dec 88


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