pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins.

A new adsorbent based on pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles was developed for specific recognition and efficient adsorption of proteins. Arising from the synergistic effect of specific affinity of apatamer on protein and tunable hydrophobic/hydrophilic property of pH-responsive polymer, the adsorbent exhibited excellent adsorption capacity for target protein. Notably, because of the pH-responsive property of the polymer, the adsorption and desorption process could be regulated through varying environmental pH. The resultant adsorbent that immobilized with lysozyme binding aptamer was successfully applied in specific recognition and efficient adsorption of lysozyme in egg white samples and good recovery results in the range of 95.2-103.2% were obtained. Moreover, the adsorbent immobilized with cytochrome C binding aptamer also exhibited satisfactory adsorption to cytochrome C. The synergistic effect of pH-responsive polymer and aptamer promoted the recognition selectivity and adsorption capacity to target protein, illustrating a facile way for construction of more specific protein adsorbents.

An easy and rapid separation method for five major proteins from egg white: Successive extraction and MALDI-TOF-MS identification.

Five major proteins from egg white were separated using a successive extraction/precipitation protocol. The yield and purity of the separated proteins were measured. The separated proteins were confirmed by MALDI-TOF-MS, and their structures were characterized by CD spectrum. Lysozyme was first separated using FPC 3500 resin and then ovomucin from the lysozyme-free egg white. Ammonium sulfate and citric acid were added to the resulting lysozyme- and ovomucin-free egg white solution to precipitate ovotransferrin. Ovomucoid and ovalbumin were separated from the resulting supernatant using ethanol. The separated proteins were further purified and the optimal conditions for the further purifications were suggested. The purity and yield of lysozyme, ovotransferrin, ovalbumin, and ovomucoid were higher than 90% and 77%, while those of ovomucin were about 72% and 75%, respectively. This study separated five major proteins in egg white successively using resin adsorption, pH adjustment, salt/ethanol precipitation, and ultrafiltration.

Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses.

Cross-linking mass spectrometry draws structural information from covalently linked peptide pairs. When these links do not match to previous structural models, they may indicate changes in protein conformation. Unfortunately, such links can also be the result of experimental error or artifacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing coelution of the corresponding linear peptides in MS1 spectra, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress noncovalent peptide formations, increasingly disruptive ionization settings can be used, such as in-source fragmentation.

Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins.

In light of the increasing threat of bacterial drug resistance to human health on a global scale, research and development of antimicrobial peptides as a novel class of potent antibiotics has gained considerable attention. The present study focuses on the structural evaluation and membrane interaction of two new cationic antimicrobial peptides, cOT2 and sOT2, derived from Siamese crocodile (Crocodylus siamensis) and Chinese softshell turtle (Pelodiscus sinensis) ovotransferrins. Here, cOT1 (+3) and sOT1 (+3) were derived from reptile ovotransferrins by chromatographic purification and characterized by mass spectrometry and N-terminal sequencing analysis. In order to increase the antimicrobial efficacy, two novel peptides, cOT2 (+6) and sOT2 (+5), were designed and synthesized as "naturally-engineered" by primary amino acid sequence extension of cOT1 and sOT1, respectively. These rational designs of modified peptides were assayed in term of antimicrobial activity. These peptides display strong antimicrobial activity against several bacterial strains, e.g. Vibrio cholerae, Bacillus megaterium, and Bacillus pumilus TISTR 905, with MICs of 7-16.1µM. In terms of structural conformation in mimic environments, CD spectroscopic analysis of the secondary peptides structure features revealed fairly the similarity on α-helical content with magainin II. Hence, the modes of actions have been speculated as toroidal and carpet model. Furthermore, the disruption of intact bacterial cells induced by cOT2 and sOT2 was investigated by SEM and AFM. The results provided evidence that cOT2 and sOT2 have the potential to cause different morphological changes of bacterial cells and that these effects can be enhanced by increasing the peptide concentration.

A "Turn-on-off-on" fluorescence switch based on quantum dots and gold nanoparticles for discriminative detection of ovotransferrin.

A novel strategy for ovotransferrin (OVT) detection by tracing the "on-off-on" fluorescence signals of quantum dots (QDs) and gold nanoparticles (AuNPs) utilizing fluorescence measurements were established in this article. The immune interaction between QDs-OVT and anti-OVT-AuNPs leads to drastic quenching (turning off) of the donor by a fluorescence resonance energy transfer (FRET) process. After the addition of free OVT, anti-OVT-AuNPs peel off from the QDs-OVT surface and bind to free OVT due to competitive immunoreactions, resulting in the restoration of the fluorescence intensity of QDs (turning on). Consequently, this process can be utilized for the selective detection of OVT via optical responses. Under optimal conditions, the fluorescence intensity of the system increased linearly with increasing concentrations of OVT from 0.05 to 3.8 µg/mL. The limit of detection of OVT was 23.55 ng/mL. This method was applied to the analysis of OVT in egg powder samples. Furthermore, the strategy provided in this work could be conveniently followed to establish similar immunosensors for the rapid detection of other proteins with corresponding antibodies.

Aggregation of egg white proteins with pulsed electric fields and thermal processes.

BACKGROUND: Pulsed electric field (PEF) processing is progressing towards application for liquid egg to ensure microbial safety. However, it usually causes protein aggregation, and the mechanism is still unclear. In this study, egg white protein was applied to investigate the changes in protein structure and mechanism of aggregates formation and a comparison was made with thermal treatment. RESULTS: Soluble protein content decreased with the increase of turbidity after both treatments. Fluorescence intensity and free sulfhydryl content were increased after being treated at 70 °C for 4 min. Less-remarkable changes of hydrophobicity were observed after PEF treatments (30 kV cm(-1) , 800 µs). Soluble and insoluble aggregates were observed by thermal treatment, and disulfide bonds were the main binding forces. The main components of insoluble aggregates formed by thermal treatment were ovotransferrin (30.58%), lysozyme (18.47%) and ovalbumin (14.20%). While only insoluble aggregates were detected during PEF processes, which consists of ovotransferrin (11.86%), lysozyme (21.11%) and ovalbumin (31.07%). Electrostatic interaction played a very important role in the aggregates formation. CONCLUSION: PEF had a minor impact on the structure of egg white protein. PEF had insignificant influence on heat-sensitive protein, indicating that PEF has potential in processing food with high biological activity and heat sensitive properties. © 2015 Society of Chemical Industry.

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid chromatography with a single column (2DLC-1C), which can also be employed to separate three kinds of active proteins completely, such as lysozyme, ovotransferrin and ovalbumin from egg white. The result is very important not only to the development of new 2DLC technology with a single column for proteomics, but also to recombinant protein drug production for saving column expense and simplifying the process in biotechnology.

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers.

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell's thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality.

Antimicrobial activity of cuticle and outer eggshell protein extracts from three species of domestic birds.

1. The eggshell cuticle is the proteinaceous outermost layer of the eggshell which regulates water exchange and protects against entry of micro-organisms. In this study, we investigated the hypothesis that the cuticle may also reduce microbial contamination by providing a chemical defence. 2. Outer eggshell and cuticle protein was extracted from domestic chicken (Gallus gallus), duck (Anas platyrhynchos) and goose (Anser anser) eggs by HCl and urea treatment, respectively. Antimicrobial activity of the extracts against Gram-positive and Gram-negative bacteria was evaluated. 3. C-type lysozyme, ovotransferrin and ovocalyxin-32 were identified in all extracts by Western blotting. All extracts from all species demonstrated lysozyme enzymatic activity. Immobilised c-type lysozyme retained some enzymatic activity. Protein extracts demonstrated activity against Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis suggesting the action of antimicrobial proteins in addition to lysozyme. 4. The results suggest that the antimicrobial outer eggshell and cuticle proteins present in a number of avian species may be a mechanism which enhances avian reproductive success.

Comparative antibacterial activity of avian egg white protein extracts.

1. Egg white proteins from the eggs of domestic chicken (Gallus gallus), turkey (Meleagris gallopavo), duck (Anas platyrhynchos) and goose (Anser anser) were analysed in order to compare the antimicrobial activity of these products. 2. Albumen from each species was sampled and analysed by SDS-PAGE and Western blotting. Antimicrobial activity and lysozyme activity were measured. 3. Ovotransferrin and ovalbumin were identified in all species while c-type lysozyme was present in chicken, turkey and duck egg white samples, but not in goose. 4. Galliformes appear to possess albumens with greater antimicrobial activity than those of the Anseriformes. This can be attributed to higher concentrations of ovotransferrin and the broad acting c-type lysozyme.

Estimations of repeatability and heritability of egg albumen antimicrobial activity and of lysozyme and ovotransferrin concentrations.

1. The repeatability and heritability of growth inhibition by egg albumen of two major pathogenic bacteria, a Gram-negative (Salmonella Enteritidis) and a Gram-positive (Staphyloccocus aureus) and of two antimicrobial albumen proteins, lysozyme and ovotransferrin, were estimated in commercial pedigree hens. 2. Repeatability was evaluated in 100 egg-type hens at the beginning, middle and end of the laying cycle on eggs collected for 3 weeks. Heritabilities were estimated at 36 to 40 weeks of age on 400 pedigree hens (2 eggs/hen), which were the offspring of 25 sires each mated with 4 dams. Ovotransferrin and lysozyme were quantified by ELISA. Salmonella Enteritidis (S.E.) and Staphyloccocus aureus (S.A.) were inoculated into a sample of sterilised albumen and enumerated after incubation. 3. Total protein content in albumen decreased with age of laying hens, whereas there were increases in lysozyme or ovotransferrin concentrations and in the bacteriostatic effect of albumen. 4. Repeatability for bacterial growth in albumen ranged from 0.29 to 0.39 for the number of S.E. (log cfu/ml) one day post inoculation (p.i.) but was lower and more variable at 5 d p.i. or for S.A. number. It ranged from 0.27 to 0.38 for S.E. and S.A. number at the mid period of the laying cycle. Repeatabilities were low and variable for total egg albumen protein or lysozyme and ovotranferrin concentrations (0 to 0.22). 5. Negative phenotypic correlations were observed between lysozyme concentrations and S.E. number but that between lysozyme and S.A. number was not significant. 6. Heritabilities were low (0.01 to 0.09) for protein traits. They were 0.11 for S.A. number and 0.16 for S.E. number one day p.i. 7. It appears to be more efficient to select on global bacterial growth than on specific antimicrobial proteins. The most promising trait is the number of S.E. one day p.i.

Identification of eggshell membrane proteins and purification of ovotransferrin and beta-NAGase from hen egg white.

Exposure of selected Gram-positive and Gram-negative bacterial pathogens to egg shell membranes (ESM) significantly reduced their thermal resistance and/or inactivated cells. Although the components responsible for this antibacterial activity have not been conclusively identified, several proteins associated with the ESM activity have been identified including beta-N-acetylglucosaminidase, lysozyme and ovotransferrin, with each displaying varying degrees of antibacterial activity. Numerous attempts to purify active fractions of beta-N-acetylglucosaminidase, lysozyme and ovotransferrin from the ESM proved somewhat limited; however, hen egg white (HEW) beta-N-acetylglucosaminidase was purified using a two-step chromatographic procedure, isoelectric focusing followed by cation exchange chromatography. Pure fractions of ovotransferrin were also obtained in the process. SDS-PAGE electrophoresis and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry were then used to partially characterize the individual protein components. Purified protein fractions such as these will be required in order to fully elucidate the mechanism responsible for the antimicrobial properties associated with the ESM.

Improved detection of higher molecular weight proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on polytetrafluoroethylene surfaces.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins was performed on a range of polytetrafluoroethylene (PTFE) surfaces. Sinapinic acid and alpha-cyano-4-hydroxycinnamic acid matrices were compared and the order of application varied to identify the best combination for each surface. It is demonstrated that the use of a PTFE surface improves the intensity of signals obtained for higher molecular weight proteins.

Producing a low ovomucoid egg white preparation by precipitation with aqueous ethanol.

A novel method for producing a low ovomucoid egg white preparation is proposed. Egg white powder (0.5 g) was dissolved in a 10-fold weight of distilled water and adjusted to pH 5, and ethanol was added to the solution at a final concentration of 20% (v/v). The mixture was vigorously stirred and centrifuged. The precipitate was washed three times with 20% ethanol (6.25 ml each), with about 65% of egg white proteins occurring in the precipitate. The use of ELISA demonstrated that 70% of ovomucoid was recovered from the supernatant fraction. However, functionally important proteins such as ovalbumin, ovotransferrin, and lysozyme still remained in the precipitate. These results may be due primarily to the much higher solubility of ovomucoid in this aqueous ethanol. Food quality evaluation showed that high whippability and foam stability were retained in the low ovomucoid preparation as in its material egg white. This product would thus be applicable as a new processed food for ovomucoid-sensitive allergic patients.

The effect of avian uterine fluid on the growth behavior of calcite crystals.

Eggshell formation takes place on the eggshell membrane in an acellular medium, the uterine fluid that contains the inorganic minerals and precursors of the organic matrix. The high degree of eggshell structure could be due to an interaction between calcium carbonate and the organic matrix. The aim of this study was to demonstrate such an interaction by measuring the effect in vitro of uterine fluid collected at various phases of shell formation on precipitation kinetics, size, and morphology of calcite crystals. The SDS-PAGE profiles of the organic constituents differed between the different phases of eggshell formation. The predominant constituents were ovalbumin and ovotransferrin at the initial phase and lysozyme, ovocleidin-17, ovocalyxin-32, 36- and 21-kDa bands, and ovocleidin-116 at the growth phase. These proteins were numerous in the terminal phase and showed an increased staining of the 32- and 66-kDa bands and appearance of very low molecular weight bands. The precipitation lag time was shortened in proportion to the protein concentration at the initial stage. The effect was observed with a lower magnitude in the presence of constituents of growth and terminal phases. Crystal size was smaller in the presence of constituents from the three stages compared with the control. Components from the initial phase induced the formation of twinned crystals and of rounded corners in the rhombohedric crystals. The presence of components from the growth and terminal phases strongly modified the morphology of the calcite crystals. The majority of the corners became rough and developed curved faces. These observations confirm the interaction of the uterine fluid with calcite and its contribution to eggshell structure.

Extraction and quantification by ELISA of eggshell organic matrix proteins (ovocleidin-17, ovalbumin, ovotransferrin) in shell from young and old hens.

The eggshell matrix is mainly composed of proteins that are thought to influence shell formation and calcification and, thus, modify the resulting properties of the shell. We investigated the potential of some of these proteins as biomarkers of eggshell quality by developing a competitive indirect ELISA for quantifying ovotransferrin, ovalbumin, and ovocleidin-17 in eggshell extract. Eggshell fragments were demineralized in acetic acid (20%) and freeze-dried. The micro-extraction yield was markedly increased (>50%) when Tween 20 was added to the subsequent extraction and dialysis milieus. Microplates were coated with ovotransferrin and ovalbumin in a 0.1M carbonate-bicarbonate buffer, but ovocleidin-17 was fixed with acetone (-20 C, 20 min). Optimal dilutions of the monoclonal (ovotransferrin) and polyclonal (ovalbumin and ovocleidin-17) antibodies were 1/3,000, 1/25,000 and 1/4,000, respectively. The inhibition curves were optimized by preincubating the antibodies and proteins overnight. The intraassay coefficient (<5%), parallelism of the standards and samples curves, and recovery (101%) were satisfactory for ovotransferrin. Measurements of ovalbumin were less precise because of higher interassay variation and differences between the slopes of standard and sample inhibition curves. Ovocleidin-17 assays showed similar slopes for standard and eggshell extracts. Although the total protein in soluble matrix extracts was not affected by age, the concentrations of these proteins were higher in eggshell extracts from older hens compared with those from young hens: 1.98x for ovotransferrin, 1.86x for ovalbumin, and 1.58x for ovocleidin-17. The quantification of specific eggshell matrix proteins in shell of differing quality is, therefore, a promising tool for analyzing the origin of eggshell faults and may provide useful information for breeding programs.

Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.

We report a method for the analysis of picomolar concentration proteins using electrophoretically mediated microanalysis (EMMA) to label proteins on-column with a fluorogenic reagent. Labeling is followed by capillary zone electrophoresis separation and postcolumn detection based on laser-induced fluorescence. The method provides a concentration detection limit (3 sigma) of 3 x 10(-13) M for conalbumin. The method provides separation efficiency of 300,000 theoretical plates. Protein extract from a human colon adenocarcinoma cell line generated a dozen major components and many minor components in a 12-min separation; the protein extract from 2.5 cells was used for this analysis. When compared to UV absorbance detection, the EMMA method provides 7,000,000-fold improvement in detection limit.

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run-to-run migration times.

Metalloprotein analysis by capillary isoelectric focusing.

Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were employed that generated linear pH gradients of 3-10 and 5-8. Several different proteins and one peptide with known isoelectric points (pIs) were used to establish linear relationships between peak migration time and pI. These standards were also used as internal markers to estimate peak pI values of the metalloproteins subjected to cIEF. Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3-10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe3+/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3-10 gave a similar separation pattern to conalbumin with four major peaks at pI values of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (diferric forms). Additional resolution of the molecular forms of both conalbumin and transferrin was achieved using a narrower pH gradient (5-8). Rabbit liver MT subjected to cIEF with a pH gradient of 3-10 gave a complex separation pattern with two prominent peaks (pI values of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. When individual MT isoforms (MT-1 and MT-2) were separately subjected to cIEF with a pH gradient of 3-10, heterogeneous peaks with higher pI values (4.12-4.74) were observed. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. MT samples could be separated using a pH gradient of 5-8 despite the fact that their apparent pI values were below the limits of the pH gradient established. In general, the heterogeneity observed for conalbumin, transferrin and MT proteins subjected to cIEF reflects the presence or absence of bound metal. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.

Development of a high-performance capillary isoelectric focusing technique with application to studies of microheterogeneity in chicken conalbumin.

A robust, simple, reproducible isoelectric focusing method using capillary electrophoresis that exhibits high stability, migration time reproducibility and pH linearity over a wide pH gradient was developed. Consecutive runs (over 113 runs) of several proteins and one peptide with isoelectric points (p/s) ranging from 9.45 to 2.75 yielded excellent migration time reproducibility (< 2% R.S.D.). Experimental parameters including buffer aging and capillary-to-capillary variation were thoroughly examined and optimized to improve the migration time reproducibility. The capillary isoelectric focusing (CIEF) method was applied to the analysis of chicken conalbumin (ovotransferrin), an iron-binding protein in egg white. Conalbumin (low iron content) separated into three major components with p/s of 7.2, 6.6 and 6.2. When the protein was saturated with iron (2 Fe/mol), a shift to lower p/s was detected. Chicken serum transferrin subjected to CIEF gave a pattern similar to conalbumin with three p/s of 7.1, 6.6 and 6.1, indicating that it was not fully saturated with iron. Thus, CIEF can be used as a potential analytical method to provide information about the metal-binding properties of specific metalloproteins.