Hen Egg White Research Articles

Investigation of lysozyme adsorption performance of Cu2+-attached PHEMA beads embedded cryogel membranes

For the purification of large molecules, cryogels are an alternative stationary phase to particle based media. But, due to the drawbacks of cryogels (i.e., low surface area) and particle sorbents (i.e., pressure drop in fixed beds), in recent years, the use of hybrid cryogels has greatly increased. In this study, a novel hybrid cryogel column was synthesized for the purification of lysozyme from aqueous solutions and hen egg white (HEW). Firstly, poly(2-hydroxyethyl methacrylate) beads (2μm size) were prepared, and after iminodiacetic acid (IDA) immobilization, Cu2+ ions were attached via the IDA chelating groups. These arranged Cu2+-attached PHEMA beads (Cu2+-ABs) were located into PHEMA based cryogel in order to prepare Cu2+-ABs embedded supermacroporous hybrid cryogel (Cu2+-ABsEHC) column. The specific surface area of the hybrid cryogel was found as 95m2/g by using BET. The amount of IDA on beads was found as 875μmol IDA/g. It was approached to the optimum adsorption levels at initial lysozyme concentration of 4mg/mL and pH8.0 as 874.9mg/g beads. The purity of lysozyme adsorbed from HEW was studied by SDS-PAGE with a purity of 86.4%. It is demonstrated that this column is a potential separation medium for purification of lysozyme from HEW.

Modeling and control of shape distribution of protein crystal aggregates

We focus on the modeling and control of protein crystal aggregates in a large-scale batch crystallizer used to produce tetragonal hen-egg-white (HEW) lysozyme crystals. We initially present a kinetic Monte Carlo (kMC) simulation for the modeling of the crystal nucleation, growth, and shear-induced aggregation in an effort to control the evolution of the crystal shape distribution. Through experimental data, the crystal growth rate is calibrated and an empirical expression for the nucleation rate is also developed. Then, the method of moments is applied to a comprehensive population balance model to derive a reduced-order moment model that describes the dynamic evolution of the three leading moments of the crystal volume distribution. Along with mass and energy balances for the continuous phase, the moment model is used to design a model predictive control (MPC) strategy which drives the crystal shape distribution to a desired set-point value through the manipulation of the crystallizer jacket temperature. Compared to conventional operating strategies used in industry, it is demonstrated that the proposed MPC strategy is able to produce crystal aggregates with a desired shape distribution and a low polydispersity effectively dealing with the undesired effects of biased nucleation, depletion in the solute concentration, and changes in the average crystal shape due to the aggregation process.

Pilot-scale separation of lysozyme from hen egg white by integrating aqueous two-phase partitioning and membrane separation processes

A novel, cost-effective method of lysozyme separation from hen egg white was studied. This method integrates aqueous two-phase partitioning in the system EO50PO50/phosphates with membrane separation processes. The experiments were carried out in a pilot-scale on crude hen egg white.Initially, by forming an aqueous two-phase system, lysozyme was selectively extracted to the upper, polymer-rich phase while the other egg white proteins partitioned to the lower, phosphate-rich phase. Then, in order to recover lysozyme, thermoseparation of polymer-rich phase was applied. A novel approach for the simultaneous thermoseparation of the polymer-rich phase as well as for the recovery of the lysozyme was proposed, using a cross-flow microfiltration. Additionally, recovery of proteins by ultrafiltration from lower, phosphate-rich phase was also investigated.Lysozyme could be obtained after the thermal phase separation by means of microfiltration at a total recovery over the extraction steps of 47.5 and the purification factor of 10.5. The specific activity of lysozyme preparations was 34 188U/mg of protein. Using cross-flow membrane techniques, it was found that the recovery of the polymer by microfiltration from the top phase was 83.9.

Resolution enhancement in hydrophobic interaction chromatography via electrostatic interactions

Abstract In this work, a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bonding β-phenylethylamine as a functional ligand to silica. This stationary phase can separate proteins with similar hydrophobicity that traditional hydrophobic resins cannot. Hen egg white was separated to examine the selectivity. The results show that the introduced electrostatic interactions are an important factor for the resolution enhancement and the new resin could have important applications in separation and purification of biological macromolecules.

Fast separation of hen egg white protein with a phosphorylcholine type zwitterionic ion chromatography stationary phase

Abstract In this work, a kind of preparation method of zwitterionic ion chromatography (ZIC) stationary phase modified with phosphorylcholine (PC) was obtained by hydrolyzing after bonding phosphorylcholine dichloride to diol-silica to better explore the characteristics of the PC groups as ZIC stationary phase ligand in simultaneous separation of acidic proteins and basic proteins. The results showed that two kinds of acidic proteins and three kinds of basic proteins can be separated completely, meanwhile, hen egg white was separated and purified and three kinds of egg white components ovalbumin, G2 ovoglobulin and ovotransfemin proteins were separated completely by one single step on PC-ZIC column, the purity of all proteins reached above 95%. PC-ZIC stationary phase was successfully improved with better separation capacity and selectivity than previously reported in this paper.

Large-Scale Production of Microcrystals and Precipitates of Proteins and Their Complexes

The optimum conditions for the formation of plate-like and urchin-like microcrystals of biomolecules and their transfer to rotors for solid-state NMR spectroscopy depend on a variety of factors, of which minimizing the manipulation of the microcrystals and storing the sample for several months at 277 K (4 °C) play an important role. Three biological systems were investigated: Hen Egg-White (HEW) lysozyme (129 residues), the lengthened C-terminal domain (LCter) of Human centrin 2 (89 residues), and the complex between the C-terminal domain (Cter) of Human centrin 2 (79 residues) and the P17-XPC peptide (17 residues).

Oral Immunotherapy for Pollen Allergy Using T-Cell Epitope-Containing Egg White Derived from Genetically Manipulated Chickens

Peptide immunotherapy using T-cell epitopes is expected to be an effective treatment for allergic diseases such as Japanese cedar (Cryptomeria japonica; Cj) pollinosis. To develop a treatment for pollen allergy by inducing oral tolerance, we generated genetically manipulated (GM) chickens by retroviral gene transduction, to produce a fusion protein of chicken egg white lysozyme and a peptide derived from seven dominant human T-cell epitopes of Japanese cedar pollen allergens (cLys-7crp). The transgene sequence was detected in all chickens transduced with the retroviral vector. Transduction efficiency in blood cells correlated to transgene expression. Western blot analysis revealed that cLys-7crp was expressed in the egg white of GM hens. Mice induced to develop allergic rhinitis by Cj pollinosis were fed with cLys-7crp-containing egg white produced by GM chickens. Total and Cj allergen (Cry j 1)-specific IgE levels were significantly decreased in allergic mice fed with cLys-7crp-containing egg white compared with allergic mice fed with normal egg white. These results suggest that oral administration of T-cell epitope-containing egg white derived from GM chickens is effective for the induction of immune tolerance as an allergy therapy.

Structure and ACE-Inhibitory Activity of Peptides Derived from Hen Egg White Lysozyme

Angiotensin I-converting enzyme plays an important role in hypertension and therefore its inhibition is considered to be a useful procedure in the prevention of hypertension. Two novel ACE inhibitory peptides were purified and identified from the papain-trypsin hydrolysate of hen egg white lysozyme using reverse phase-high performance liquid chromatography. The sequences of identified peptides were NTDGSTDYGILQINSR (MW: 1,753.98 ± 0.5 Da) and VFGR (MW: 459.26 ± 0.5 Da), which were named F2 and F9 peptide, respectively. Analyses of the far-UV CD spectra of ACE in the absence and presence of the F2 peptide revealed ACE secondary structural changes. In the presence of the F2 peptide, a loss of helical content of ACE was observed, which can lead to decrease of the enzymatic activity. Lineweaver–Burk plots show that the identified peptides both act as non-competitive ACE inhibitors. These findings would be helpful on the understanding of interaction between ACE and its inhibitory peptides.

Crowding versus molecular seeding: NMR studies of protein aggregation in hen egg white

In living systems, proteins are surrounded by many other macromolecules of different nature, at high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, such as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides or even synthetic polymers. We have recently proposed hen egg white (HEW) as a suitable, natural medium to study macromolecules in crowding conditions. Here, we show that HEW can increase dramatically the aggregation kinetics of proteins with an in-built tendency to associate. By dissecting the mechanism we demonstrate that only part of this effect is due to crowding, while another factor playing an important role is the interaction with proteins from the milieu. High molecular weight glycoproteins present in HEW act as efficient molecular seeds for aggregation. Our results bear important consequences for in-cell NMR studies and suggest a role of glycosylated proteins in aggregation.

Polydopamine-graft-PEG antifouling coating for quantitative analysis of food proteins by CE

In this paper, firstly the surface plasma resonance (SPR) was used to evaluate the antifouling property of polydopamine-graft-poly(ethylene glycol) (PEG) copolymer coating, and then the quantitative analysis of food proteins (such as hen eggs and milk powder) was investigated by capillary electrophoresis (CE) method using polydopamine-graft-PEG copolymer coated capillary. The great separations of lysozyme, conalbumin and ovalbumin from hen egg white could be achieved in phosphate–citrate buffer (pH 3.20) with an applied voltage of +20 kV in polydopamine-graft-PEG copolymer coated capillary. The range of protein recovery percentage of the three kinds of egg white proteins was 96.67% to 101.02%. According to the quantitative analysis results, the protein content of lysozyme, conalbumin and ovalbumin in hen egg white was 13.268 mg mL−1, 28.360 mg mL−1 and 157.418 mg mL−1, respectively. Whey proteins (β-lactoglobulin A, β-lactoglobulin B and α-lactalbumin) in nonfat milk powder could be separated completely with an applied voltage of −20 kV in sodium tetraborate buffer (pH 8.5). The protein recovery of β-lactoglobulin A, β-lactoglobulin B and α-lactalbumin was in the range of 86.40–94.38%. Finally, the quantitative protein content of β-lactoglobulin A, β-lactoglobulin B and α-lactalbumin in nonfat milk powder was 1.418 mg mL−1, 0.129 mg mL−1, and 0.073 mg mL−1, respectively. Also the egg white proteins could be separated consecutively 25 times without a noticeable loss in separation efficiency using polydopamine-graft-PEG copolymer coated capillary in one day.

Mobility of Molecules and Ions Solubilized in Protein Gels: Diffusion in the Thick Fraction of Hen Egg White

The thick fraction of hen egg white is a protein hydrogel with an immeasurably high viscosity composed of ∼90% water that can serve as a model system for mammalian mucous membrane. Measurements of the rate constants of diffusion-controlled reactions occurring within the gel (and corresponding activation energies) and electric conductivity revealed that the thick fraction of egg white can be envisioned as a 3D network comprising hydrated protein molecules (held by intermolecular S-S bridges) surrounded by water pools and channels (of nonuniform diameters) that have a microviscosity that is very similar to that of bulk water. This was corroborated by differential scanning calorimetry measurements that revealed that 16% of water is bound to proteins. The melting kinetics of ice crystallites (produced from the freezable water) indicates nonhomogeneous water pool size.

N-glycosylation of ovomucin from hen egg white

Ovomucin is a bioactive egg white glycoprotein responsible for the gel properties of fresh egg white and is believed to be involved in egg white thinning, a natural process that occurs during storage. Ovomucin is composed of two subunits: a carbohydrate-rich β-ovomucin with molecular weight of 400-610KDa and a carbohydrate-poor α-ovomucin with molecular mass of 254KDa. In addition to limited information on O-linked glycans of ovomucin, there is no study on either the N-glycan structures or the N-glycosylation sites. The purpose of the present study was to characterize the N-glycosylation of ovomucin from fresh eggs using nano LC ESI-MS, MS/MS and MALDI MS. Our results showed the presence of N-linked glycans on both glycoproteins. We found 18 potential N-glycosylation sites in α-ovomucin. 15 sites were glycosylated, one site was found in both glycosylated and non-glycosylated forms and two potential glycosylation sites were found unoccupied. The N-glycans of α-ovomucin found on the glycosylation sites are complex-type structures with bisecting N-acetylglucosamine. MALDI MS of the N-glycans released from α-ovomucin by PNGase F revealed that the most abundant glycan structure is a bisected type of composition GlcNAc(6)Man(3). Two N-glycosylated sites were found in β-ovomucin.

A natural and readily available crowding agent: NMR studies of proteins in hen egg white

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media. Proteins 2011. © 2010 Wiley-Liss, Inc.

Ultra-sound assisted formation of biodegradable double emulsion capsules from hen egg white

It was found that directed self-assembly of hen egg white proteins at oil–water interfaces in Pickering emulsions followed by acoustic denaturation of the proteins at the interface produces double emulsion capsules. The formation of capsules is dependent on the pH of the aqueous phase and on the concentration. At pH 5.5, the capsules are easily formed while at pH 8.5 the formation is difficult and only occurs at high concentration and in low quantity. The difference in capsule stability and interface stabilization at different pH was rationalized by competitive stabilization forces at the oil–water interface which was investigated by dynamic interfacial tension measurements. While the absolute interfacial stabilization does not differ much at different pH, when taking into account stabilization behavior of the pure buffer solutions without protein there is a significant difference. At neutral and alkaline conditions we observed that the pure buffer already stabilizes the interface which competes with the stabilization by the protein. This competitive stabilization resulted in less stable capsules formed at higher pH. Moreover, a lower pH enhances the ultra-sound assisted denaturation of the proteins, yielding an additional stabilization of the formed structures. In addition, the capsules are biodegradable. Upon digestion with trypsin, they were completely destroyed leaving behind small protein aggregates.

Suggestion for New Protocol to Remove Hypermelanosis Using Lysosome Related Enzymes Extracted from Hen's Egg White

Suggestion for New Protocol to Remove Hypermelanosis Using Lysosome Related Enzymes Extracted from Hen's Egg White

Role of the Electric Double Layer in Controlling the Nucleation Rate for Tetragonal Hen Egg White Lysozyme Crystals by Application of an External Electric Field

The nucleation rate of tetragonal hen egg white (HEW) lysozyme crystals could be increased or decreased via application of an ac electric field, which modifies the chemical potential of the liquid and solid states. The strength of an external electric field necessary to control the nucleation rate was calculated to be 104−105 V/cm, which was larger than that used experimentally by 2 or 3 orders of magnitude. This huge discrepancy could be understood by assuming the presence of a very large electric field sustained in the electric double layer (EDL) that forms at the interface between the dissimilar phases. By changing the frequency of an applied electric field of 800 V/cm in the range of 1−9 MHz, we were able to increase or decrease the nucleation rate. Specifically, the nucleation rate with an applied electric field increased with an increasing applied frequency up to 5 MHz and then significantly dropped above this value. The rapid decrease is considered to result from extinction of the EDL above 5 MHz.

Hen eggwhite-mediated stack crystallization of calcium carbonate

In this paper, the stack-like crystallization of calcium carbonate in the presence of hen eggwhite under direct drying and vacuum freeze drying was investigated, and marked morphological changes in the calcium carbonate particles were observed depending on the reaction condition used. Scanning electron microscopy (SEM), Powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), and Nano Mechanical Tester were employed to characterize the samples. Results indicate that gelling eggwhite-mediated the formation of the “stack-like” layered calcium carbonate aggregates composed of considerable nanosheets under direct drying while only rhombohedra calcite crystal (1 0 4) was formed without any additives. An analogous structure to the brick-and-mortar arrangement was attainted by vacuum freeze drying. The average elastic modulus and the hardness of “stack-like” calcium carbonate hybrid material were assessed 0.9952 and 0.0415 GPa with Nano-indenter test, respectively.

Oligomerisation, Fibrillation and Activity of Hen Lysozyme in Alkaline Medium: A Concentration Dependent Investigation

Oligomerisation and fibrillation are hallmarks of protein misfolding diseases like Alzheimer's, Prion, Parkinson's, systemic amyloidosis and so on. Hen eggwhite lysozyme (HEWL) which spontaneously aggregates at pH 12.2 under room temperature as shown by us previously is an excellent model protein for aggregation studies. The present work was focussed on investigating the role of protein monomer concentration on the size and morphology of aggregates. For this purpose we employed HEWL concentrations ranging from 120 μM to 300 nM. Our findings reveal that A) FRET efficiency between dansyl labelled HEWL and dabcyl labelled HEWL in the aggregate, monitored over 12 hours was inversely proportional to HEWL monomer concentration B) Size exclusion chromatography after 12 hours showed that while all aggregates of 50 & 120 uM HEWL eluted much later, a small population of aggregates from 10 uM HEWL eluted early through the void volume C) Binding of ANS revealed fluorescence spectra that were gradually blue shifted and more intense, over 12h, in the following order with aggregates of 120 > 50 > 3 uM, while for 300 nM, these spectra were fairly invariant in emission wavelength and intensity over 12 hours. D) HEWL enzymatic activity decreased almost uniformly with time in alkaline pH for all concentrations suggesting a concentration independent early unfolding step E) AFM images showed extensive fibrils in 3 & 0.3 μM HEWL within 12 hours but predominantly large globular aggregates with 120 μM and 50 μM HEWL samples in the same time period. The above results clearly suggest that size and morphology of HEWL aggregates at alkaline pH are critically dependent on the initial monomer concentration.

Modelling protein crystallisation using morphological population balance models

Protein crystallisation is known to be affected by many factors and inherently difficult to control. Being able to model the crystal growth behaviour, especially at process scale for the population of particles in a crystalliser will no doubt greatly help the formulation and controlled manufacture of protein crystals. In this paper, a morphological population balance model for crystallisation of tetragonal Hen-Egg-White (HEW) lysozyme is presented. Since the population balance model has incorporated crystal shape information, it is able to simulate the dynamic evolution of the shape distribution as well as size distribution. The morphological population balance model requires faceted growth kinetics data, which was obtained from published data in literature for the two identified independent crystallographic faces, {1 0 1} and {1 1 0}, of HEW lysozyme crystals.

Elastic constants in tetragonal hen egg-white lysozyme crystals containing large amount of water

Transverse sound velocity of cross-linked tetragonal hen egg-white (HEW) lysozyme crystals containing large amount of water in the crystal was measured using ultrasonic pulse-echo method. All elastic constants of cross-linked crystals were observed to be C11=C22=5.50 GPa, C12=4.33 GPa, C13=C23=3.94 GPa, C33=5.22 GPa, C44=C55=0.68 GPa, and C66=0.84 GPa, respectively. We found that the elastic constants of the cross-linked crystals are identical to those of the intrinsic ones without cross-linking. Moreover, we found that tetragonal HEW lysozyme crystals that enclose large amount of water show decreased elastic constants (softening). In particular, the shear elastic constants C44=C55 and C66 showed more softening effect comparing with other elastic components.