Chicken Egg White Research Articles

Antibacterial activity of lysozyme-binding proteins from chicken egg white

The purpose of this study was to establish a method for determining the bacteriolytic activity after separation of lysozyme-binding proteins from egg white. Lysozyme-binding proteins such as ovotransferrin and ovalbumin were separated by non-denaturing two-dimensional electrophoresis (2DE) and transferred to a membrane. The lysozyme activity of the separated and immobilized egg white proteins was assessed directly to produce a non-denaturing 3D map of the egg white proteins by incorporating an axis that combined each spot's lysozyme-activity with the non-denaturing 2DE pattern. Lysozyme-ovotransferrin and lysozyme-ovalbumin complexes could be reconstructed in vitro after the cathode end fraction containing lysozyme was added to purified ovotransferrin and ovalbumin, respectively. These complexes retained lysozyme activity even after separation by non-denaturing 2DE. Furthermore, when the lysozyme-ovotransferrin complex from egg white was extracted after separation by isoelectric focusing by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, the complex possessed bacteriolytic activity against both Bacillus subtilis and Escherichia coli. These methods can be applied to investigate protein complexes possessing bacteriolytic activity against a wide range of both Gram-positive and Gram-negative bacteria.

Chicken egg white and L-cysteine as cooperative ligands for effective encapsulation of Zn-doped silver nanoclusters for sensing and imaging applications

Herein, we report chicken egg white (CEW) and L-cysteine (L-Cys) as cooperative ligands for effective encapsulation of Zn-doped silver nanoclusters (Zn-doped Ag NCs) for enhancing optical properties in sensing of Hg2+ ion and bioimaging of fungal cells (Alternaria sp.). The emission peak at 657 nm was remarkably enhanced by doping of Zn-atom into Ag NCs, suggesting the proteins of CEW and L-Cys provide good architectures for supramolecular encapsulation of Zn-Ag NCs. The quantum yield (QY) of Ag NC was improved to 13.3% due to the doping of Zn atoms into Ag NCs. The “fluorescence-off” of Zn-doped Ag NCs is proceeded by the addition of Hg2+ ion, which exhibits detection limit of 68 nM. The other chemical species did not interfere with assay of Hg2+ ion. We also extended the application of Zn-doped Ag NCs as a probe in imaging of fungal cells (Alternaria sp.). The CEW-L-Cys encapsulated Zn-doped Ag NCs exhibited an excellent stability and shown to be a magnificent probe for sensing and bioimaging applications, which demonstrates that Zn-doped Ag NCs as potential candidates in analytical and bioanalytical sciences.

A mass spectrometry approach for the identification and localization of lysozyme modifications by acrolein

Lipids containing polyunsaturated fatty acids are primary targets of oxidation, leading to the formation of a variety of reactive products (1). The new oxidised products are generated with different number of oxygen molecules bound to the acyl chain, increasing the lipid molecular weight. However, the alkoxyl radicals can generate short-chain oxidation products including short-chain aldehydes, which can covalently modify proteins in a process called lipoxidation (2). Acrolein, with only three carbons, is the shortest alkenal identified as a short-chain oxidation product. It is also by far the strongest electrophile and therefore the most reactive, especially with thiol groups on proteins (3). These oxidative post-translational modifications influence cell behaviour and can be involved in inflammatory diseases (4). The exact nature of many of these adducts, and their relationship with cellular effects are still unclear. There is also a need to develop sensitive mass spectrometry (MS) methods that are well-adapted for the identification of these adducts in complex biological systems. The aim of this work was to develop a mass spectrometry approach for the analysis of protein-aldehyde adducts. Lysozyme from chicken egg white was used to investigate the formation of short-chain aldehyde-containing lipoxidation products. The protein was first reduced with dithiothreitol to break disulphide bounds between cysteine residues, generating free thiols available for modification. Acrolein was then added to modify the reduced protein. The protein-aldehyde adducts formed were stabilized by reduction with sodium borohydride and identified using mass spectrometry to analyse the intact protein and tryptic digests. Analysis of intact acrolein-modified lysozyme showed that multiple sites (up to 8) could be modified. All modifications were found to be Michal adducts, increasing the protein mass by 58 Da per adduct. Analysis of tryptic digests allowed the localization of the adducts to specific amino acid residues. Six cysteine and two lysine residues were shown to be modified, which corroborates observation of up to 8 acrolein adducts per protein in the intact protein analysis. By the same proteomic approach, it was possible to identify amino acid-specific fragmentations that may be helpful in the identification of specific acrolein adducts. In summary, this study can be seen as a model testing mass spectrometry for the analysis of protein-aldehyde adducts. The combined use of direct infusion and LC-MS/MS methods helped to identify the type, the number and the location of acrolein modifications in lysozyme. This shows that MS methods provide a powerful tool for the evaluation of these modifications and further aldehydes are now being investigated. Potentially, this MS approach can be applied to discover biomarkers of adductions in cells and tissues under pathophysiological environments.

Self-assembly of natural protein and imidazole molecules on gold nanoparticles: Applications in wound healing against multi-drug resistant bacteria

Developing highly active and green antibacterial agents for pathogens, especially multidrug-resistant superbugs, is vital for solving the problem of serious antibiotic resistance. Herein, we report a unique system of gold nanoparticles coated with chicken egg white (CEW) and 2-mercapto-1-methylimidazole (MMT) as a novel antibacterial agent. The CEW was used to prepare the gold nanoparticles as a commercially available reducing and stabilizing agent, and then the MMT self-assembled on the surface of nanoparticles. The resulting Au@CEW/MMT was found to be a highly efficient antibacterial agent, and the activity is mainly attributed to the synergistic effects of MMT and Au@CEW in undermining the bacterial membrane. Meanwhile, the studies of antibacterial activities and biocompatibility of Au@CEW/MMT with different ratios of MMT conjugation to Au@CEW confirmed that Au@CEW/MMT3 (MMT:HAuCl4 = 1:50) can maintain a balance between antibacterial properties and biocompatibility. Furthermore, in an in-vivo study using the rabbit model, gauze loaded with Au@CEW/MMT3 can effectively accelerate the healing of wounds infected with methicillin-resistant S. aureus and promote the formation of collagen. Therefore, this work illustrated a promising material with broad-spectrum antibacterial activities for preclinical applications in treating wound infections.

Production of pharmaceutical proteins by transgenic animals.

Proteins are involved in a majority of the biochemical events that take place in all living organisms. Protein synthesis is directed by genes. All genes contain two major DNA regions. The region containing the genetic message proper (the 'coding region') is preceded by a regulatory region ('the promoter'), which determines when and in which organs a given gene must produce the corresponding protein. The techniques of genetic engineering allow the association of the coding region from one gene with the regulatory region from another gene. The expression of these recombinant genes may be achieved in cultured cells, in transgenic animals or in plants. This leads to the production of the corresponding proteins, including pharmaceutical proteins. Milk from transgenic animals is one potential source of pharmaceutical proteins. To achieve this, the promoters from milk protein genes are bound to DNA fragments containing the coding region of the genes of interest. The desired proteins are then taken from the milk and purified. Two human pharmaceutical proteins are on the market and about 20 projects are in development. One of the proteins produced in milk, antithrombin III, is an anticoagulant and the other, human C1-esterase inhibitor, is an anti-inflammatory. Several human proteins have been produced in the egg white of transgenic chickens and one has been approved by the United States Federal Drug Administration. This process has also been used to modify antibodies in cows. The genes that code for antibodies in the cow were deleted and replaced by human antibody genes. These cows, immunised by various antigens, then secreted purely human antibodies in their blood. Antibodies from such cows were able to attenuate the effects of Ebola virus in human patients.

The evaluation of biological value and immunochemical characteristics of the coagulated chicken egg white

The aim of the study was to investigate in vivo the biological value of the coagulated chicken egg white on growing rats and a comparative immunochemical evaluation in vitro of its antigenic power. The experiment was carried out on 50 growing Wistar male rats with a body weight of 80±5 g. The animals were randomly divided into 3 groups (n=16): control group G1 and two experimental groups G2 and G3. The animals of the control group (G1) received a basic isocaloric and isonitrogenous (20% protein of casein by caloric content) semi-synthetic diet. The animals of the experimental groups G2 and G3 received the same semi-synthetic diet in which casein was replaced by chicken egg white (CEW) and coagulated CEW, respectively. The average food intake of group G3 animals, who received the CEW coagulate, was significantly lower (13.7±0.6 g per day, p<0.05) in comparison with the control group G1 (18.4±0.6 g) and the experimental group G2 (19.2±0.5 g). Moreover, body weight gain of animals treated with coagulated CEW didn't differ significantly from the control G1 animals. Already on the 8th day of the experiment, the body weight gain of G2 animals, who consumed native CEW, was significantly higher in comparison with both other groups. The protein efficiency ratio (PER) for animals of the G3 group was significantly higher (1.96±0.04) than the values for the animals of the control group G1 receiving casein (1.49±0.05, p<0.01), and for the animals of the experimental group G2 receiving CEW (1.60±0.02, p<0.05). The results of immuneenzymatic testing of the initial antigenic power of ovalbumin in native CEW indicated that its content was 33.0% relative to the standard ovalbumin value, antigenic power of which was assumed to be 100%. The developed process of coagulation contributed to a decrease in antigenic power to 2.17%. The obtained data indicate a high biological value and low antigenic power of the coagulated CEW, which makes it prospective for the usage in the composition of food products of mass demand and specialized food products.

Analysis of color changes in chicken egg yolks and whites based on degree of thermal protein denaturation during ohmic heating and water bath treatment

Raw eggs were separated into yolks and whites before thermal treatment. An ohmic heating process at 20 kHz and 30 V was used for egg-yolk samples, and color changes were monitored with a computer vision system. Egg-white samples were treated with a water bath, and absorbance, as an expression of turbidity, was determined spectrophotometrically. Thermal protein denaturation of egg samples was analyzed by differential scanning calorimetry (DSC) using the dynamic method, and estimated for several thermal schedules by kinetics analysis. The DSC thermogram was characterized by two endothermic peaks in each sample. With increasing temperature, the yolk gradually turned from a plain orange to a vivid yellow color, while the white gradually changed from transparent to cloudy. For all samples, an accurate correlation was obtained between color changes and the non-denaturation ratio of the second peak temperature. Results are of potential value for the modeling and design of egg-based products.

Functionalized gold nanoparticles as affinity nanoprobes for multiple lectins

Glycan–lectin interactions are commonly observed in nature. Analytical methods, which are used to detect lectins that rely on the use of glycan ligand-modified nanoprobes as affinity probes, have been developed. Most of the existing methods are focused on the use of synthetic glycan ligands. Nevertheless, naturally available glycoproteins, such as ovalbumin in chicken egg whites, are good sources for fabricating glycan-immobilized nanoprobes. In this study, we generated functionalized gold nanoparticles (Au NPs) from a one-pot reaction by reacting chicken egg white (cew) proteins with aqueous tetrachloroaurate. The generated Au@cew NPs are mainly encapsulated by ovalbumin, in which the surface is decorated by abundant hybrid mannose and Galβ(1→4)GlcNAc-terminated glycan ligands. Thus, the generated Au@cew NPs containing hybrid mannose and Galβ(1→4)GlcNAc have the capability to selectively bind with their corresponding lectins. Lectins including concanavalin A, banana lectin, and ricin B that have binding moieties toward specific sugars were used as the model samples. Our results showed that the generated AuNPs can be used as multiplex affinity probes for these model lectins. Lectins can be selectively released from the Au@cew NP-lectin conjugates by using specific sugars, such as mannose, glucose, and β-lactose, as the releasing agents to release specific lectins from the conjugates. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used as the tool to characterize the released species from the nanoprobes. The limit of detection of these model lectins using the current approach was low (in nM). The feasibility of using the Au@cew NP-based affinity MALDI-MS to selectively detect specific lectins from complex samples was also demonstrated.

Chicken egg-white extracts promote OCT4 and NANOG expression and telomeres growth in 293T cells.

It will have broad applications in cell biology if one of egg cell extracts has the roles to promote cell proliferation and reprogramming. It will provide a new method for easier reprogramming somatic cells and promote cell proliferation. We found chicken egg-white extracts have roles to promote cell proliferation and reprogramming. The different ingredients were then assessed for cell proliferation activity and somatic cell reprogramming. Chicken egg-white extract ingredients that were less than 3 kDa (LT3K) promoted cell proliferation. Those ingredients that were greater than 3 kDa (GT3K) promoted the increased expression of pluripotency factors in somatic cells and promote telomeres growth in 293T cells. Chicken egg-whites can be separated into ingredients of LT3K, which act to promote cell proliferation, and GT3K, which can be used to promote somatic cell reprogramming.

Identification of N-Glycosites in Chicken Egg White Proteins Using an Omics Strategy.

Chicken egg white (CEW) is a perfect source of natural proteins that possesses outstanding functional properties and various bioactivities. The glycosylation structure of CEW proteins plays important roles in their functions, bioactivities, and allergies. The present work attempted to identify N-glycosites of CEW proteins using an omics strategy. CEW proteins were digested with trypsin and chymotrypsin; glycopeptides were enriched and deglycosylated using PNGase F in H218O water, followed by analysis using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). A total of 71 N-glycosites in 26 CEW glycoproteins were identified. Web-Logo analysis showed that most of the N-glycosites were at N-X-T (55%) and N-X-S (32%). Furthermore, two-dimensional electrophoresis of CEW clusterin demonstrated a series of spots horizontally distributed at 35-37 kDa with an extremely wide isoelectric point range of 4.54-6.68, indicating the heterogeneity of glycosylation of CEW clusterin. These results provided important information for the understanding of the structures, functions, and bioactivities of CEW glycoproteins.

In vitro cytotoxic and ACE-inhibitory activities of promod 278P hydrolysate of ovotransferrin from chicken egg white

Peptides released from egg proteins via enzymatic hydrolysis show various bioactivities such as antimicrobial, antioxidant, antihypertensive, and immunomodulatory properties. The objective of this study was to investigate the cytotoxic and Angiotensin Converting Enzyme (ACE)-inhibitory activities of ovotransferrin and its promod 278P enzyme hydrolysate. Ovotransferrin from egg white was hydrolyzed using promod 278P at 45°C for 3 hours. Using the MTT assay, the cytotoxicity of ovotransferrin and promod 278P hydrolysate of ovotransferrin were evaluated in human cancer cell lines of various tissue origins. The ACE-inhibitory activity was determined using the cleavage of a chromogenic substrate -Hip-His-Leu. The promod 278P hydrolysate of ovotransferrin showed a potent cytotoxicity (>90%) at 20 mg/mL in all cancer cell lines tested, but ovotransferrin did not. The IC50 value of the promod 278P hydrolysate of ovotransferrin against 5 different cancer cells were 10.05 ± 1.55, 3.45 ± 0.94, 4.43 ± 1.87, 4.92 ± 0.63, and 10.43 ± 3.91 mg/mL for MCF-7, HeLa, HepG2, HT-29, and LoVo cells, respectively. The promod 278P hydrolysate of ovotransferrin showed a strong ACE-inhibiting activity: at 10 mg/mL level, the hydrolysate showed 76.82 ± 1.28% inhibition to ACE-inhibitory activity, and 73.33 ± 2.56%, 56.85 ± 1.84%, 50.32 ± 3.71%, 17.30 ± 0.13%, and 4.52 ± 6.83% inhibitory activity at 5, 2.5, 1.25, 0.625, and 0.3125 mg/mL level, respectively. The IC50 value of the promod 278P hydrolysate of ovotransferrin was 1.53 ± 0.20 mg/mL. However, ovotransferrin did not show any inhibitory effect to angiotensin-converting enzyme activity. This result indicated that the promod 278P hydrolysate of ovotransferrin has a great potential as an anticancer and antihypertension agent for humans, but the information on the peptides responsible for the functional activities is not available yet.

Stable Benzacridine Pigments by Oxidative Coupling of Chlorogenic Acid with Amino Acids and Proteins: Toward Natural Product-Based Green Food Coloring.

The occasional greening of sweet potatoes and other plant tissues observed during cooking or other food processing has been shown to arise from the autoxidative coupling of chlorogenic acid (CGA, 5-caffeoylquinic acid) with amino acid components, leading to trihydroxybenzacridine pigments. To explore the potential of this reaction for food coloring, we report herein the optimized biomimetic preparation of trihydroxybenzacridine pigments from CGA and amino acids such as glycine and lysine, their straightforward purification by gel filtration chromatography, the UHPLC-MS/MS analysis of the purified pigment fraction, and a detailed characterization of the pH-dependent trihydroxybenzacridine chromophore. Similar green pigments were also obtained by analogous reaction of CGA with a low-cost protein, bovine serum albumin, and by simply adding CGA to chicken egg white (CEW) under stirring. Neither the purified pigments from amino acids nor the pigmented CEW exerted significant toxicity against two human cell lines, Caco-2 and HepG2, at doses compatible with common use in food coloring. Additions of the pure pigments or pigmented CEW to different food matrices imparted intense green hues, and the thermal stability of these preparations proved satisfactory up to 90 °C. The potential application of the greening reaction for the sensing of fish deterioration is also disclosed.

Preparation of an affinity cryogel column for lysozyme purification

ABSTRACTAffinity cryogels were synthesized using tris(hydroxymethyl)aminomethane (Tris) as ligand for specific interactions with the lysozyme (LYZ). The cryogel was produced by cryo-copolymerization at −12°C. A central composite rotational design 22 was used to optimize the immobilization procedure of the Tris on the cryogel. A maximum adsorption for LYZ (149.5 mg/g) was achieved when 376 mg/mL of Tris and 3.06 mol/L of sodium borohydride were used during the Tris immobilization. Chromatographic separation of LYZ from chicken egg white was done with a purity of 92.13%. Results showed that the affinity cryogel was a potential separation medium for LYZ purification.

Development of an affinity cryogel for one step purification of lysozyme from chicken egg white.

In this study, a supermacroporous polyacrylamide cryogel was produced by cryo-polymerization and activated with Tris(hydroxymethyl)aminomethane (Tris-cryogel) to be applied as an affinity ligand for a one step purification of lysozyme (LYZ), directly from chicken egg white (EW). The Tris-cryogel presented interconnected pores with size varying in the range of 20-80μm and swelling capacity of 19.6±0.9g/g. The axial dispersion of the Tris-cryogel was analyzed at different flow velocities and mobile phase viscosities. It was verified that higher viscosity resulted in a higher degree of dispersion, causing the HETP values to increase from 0.04cm to 0.8cm. Adsorption isotherms were measured at 15°C and 35°C at pH 7.5. A Langmuir model was fitted to the equilibrium data, with a maximum adsorptive capacity of 285mg/g at 15°C and 363mg/g at 35°C. Thermodynamic analysis based on the Van't Hoff relationship showed that the process was spontaneous and enthalpically driven. Lysozyme was purified directly from egg white in a one step purification process at different pH values (7.5, 8.5 and 9.5). Independent of the pH, the specificity of Tris-cryogel for lysozyme adsorption was confirmed. At pH 7.5, yield and purification fold were higher (30% and 45). In addition, the effect of the dilution rate on egg white and flow velocity were also analyzed and it was shown that flow velocity did not affected purification and column efficiency, and that diluting the egg white increased yield to 70% with a purification fold of 23. Results show Tris-cryogel is a promising matrix for use in high throughput purification of lysozyme from egg white.

A novel fluorimetric sensing platform for highly sensitive detection of organophosphorus pesticides by using egg white-encapsulated gold nanoclusters

Assays for organophosphorus pesticides (OPs) with high sensitivity as well as on-site screening have been urgently required to protect ecosystem and prevent disease. Herein, a novel fluorimetric sensing platform was constructed for quantitative detection of OPs via tyrosinase (TYR) enzyme-controlled quenching of gold nanoclusters (AuNCs). One-step green synthetic approach was developed for the synthesis of AuNCs by using chicken egg white (CEW) as template and stabilizer. Initially, TYR can catalyze the oxidation of dopamine to dopaminechrome, which can efficiently quench the fluorescence intensity of AuNCs at 630nm based on dynamic quenching process. However, with the presence of OPs, the activity of TYR was inhibited, resulting in the fluorescence recovery of AuNCs. This proposed fluorescence platform was demonstrated to enable rapid detection for OPs (paraoxon as model) and to provide excellent sensitivity with a detection limit of 0.1ngmL−1. Significantly, the fluorescence probe was used to prepare paper-based test strips for visual detection of OPs, which validated the excellent potential for real-time and on-site application.

Expression of recombinant human lysozyme in transgenic chicken promotes the growth of Bifidobacterium in the intestine and improves postnatal growth of chicken.

Lysozyme is one kind of antimicrobial proteins and often used as feed additive which can defend against pathogenic bacteria and enhance immune function of animals. In this study, we have injected the lentiviral vector expressing recombinant human lysozyme (rhLZ) gene into the blastoderm of chicken embryo to investigate the effect of recombinant human lysozyme on postnatal intestinal microbiota distribution and growth performance of chicken. Successfully, we generated 194 transgenic chickens identified by Southern blot with a positive transgenic rate of 24%. The average concentration of rhLZ was 29.90 ± 6.50 μg/mL in the egg white. Lysozyme in egg white of transgenic chickens had a significantly higher antibacterial activity than those of non-transgenic chickens by lysoplate assay (P < 0.05). The feces of transgenic and non-transgenic chickens were collected and five types of bacteria (Lactobacillus, Salmonella, Bifidobacterium, Staphylococcus aureus and Escherichia coli) were isolated and cultured to detect the impact of rhLZ on gut microbiota. Among the five bacteria, the number of Bifidobacterium in the intestine of those transgenic was significantly increased (P < 0.05). Moreover, the growth traits of the transgenic and non-transgenic chickens were analyzed. It was found that the 6-week shank length, 6-week weight and 18-week weight of transgenic chickens were significantly increased than that of non-transgenic chickens. The results demonstrated that rhLZ-transgenic chicken could promote the growth of Bifidobacterium in the intestine and improve the postnatal growth of chicken.

Separation of Lysozyme from Chicken Egg White by Ultrafiltration (1) - Dead-end Filtration of Egg White Solution -

Separation of Lysozyme from Chicken Egg White by Ultrafiltration (1) - Dead-end Filtration of Egg White Solution -

Facile synthesis of red emitting 3-aminophenylboronic acid functionalized copper nanoclusters for rapid, selective and highly sensitive detection of glycoproteins

As an emerging class of fluorescent probes, copper nanoclusters (Cu NCs) have been considered as an intriguing candidate for detecting biomoleculars due to their outstanding fluorescent properties, excellent biocompatibility and low cost. Herein, we fabricated bovine serum albumin (BSA) protected Cu NCs (BSA-Cu NCs) and further functionalized them with 3-aminophenylboronic acid (APBA) for selectively discerning glycoproteins. In aqueous solution, Cu2+ ions were directly reduced into BSA-Cu NCs by hydrazine hydrate (N2H4·H2O) at room-temperature using BSA as the capping agent. The synthetic process was very rapid, simple and easy for controlling due to the lack of any other complicated procedure such as heating and adjusting the pH value of the reactive mixture. The APBA-Cu NCs showed strong fluorescent emission at 630nm in the red range. So it can effectively avoid the disturbance of auto-fluorescence in biosamples. The fluorescence of the APBA-Cu NCs was obviously quenched by glycoprotein samples. Then, the APBA-Cu NCs were employed as a probe for selective capture and sensitive detection of glycoproteins with a wide linear range of 5–220nM and a low detection limit of 2.60nM owing to the covalent reaction between the boric acid group of APBA and the cis-glycol groups of the glycoproteins. The developed method was also successfully applied to determine glycoproteins in egg white of chickens and human urine samples with quantitative spike recoveries from 95% to 104%.

Adsorptive Cation Exchanger Mixed Matrix Membrane Chromatography for the Isolation of Lysozyme from Chicken Egg White

Membrane chromatography, which uses an adsorptive membrane, is an alternative technology that overcomes the limitations of packed-bed column chromatography. Mixed matrix membrane (MMM) preparation is a simple method by which adsorptive membranes can be prepared. In the current study, several potential low-cost cation exchange (CEX) resins were screened for their ability to bind lysozyme (LYS) at various pH values prior to the preparation of CEX MMMs. The best binding was shown by Lewatit CNP105 at pH 7. A subsequently prepared Lewatit CNP105—CEX MMM followed the Langmuir adsorption isotherm, with a maximum binding capacity of 223 mg LYS/g membrane. In batch binding from a chicken egg white (CEW) solution at pH 7, both positively charged (LYS, pI 10.7) and negatively charged (conalbumin (CNL), pI 6.1; and ovalbumin (OVL), pI 4.5) proteins were bound onto the MMM. However, CNL and OVL were only loosely bound and were washed out during the washing step. Almost pure LYS was recovered during the elution step, as shown by SDS–PAGE gel analysis.

Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension.

The surface hydrophobicity of a protein is an important factor for its interactions in solution and thus the outcome of its production process. Yet most of the methods are not able to evaluate the influence of these hydrophobic interactions under natural conditions. In the present work we have established a high resolution stalagmometric method for surface tension determination on a liquid handling station, which can cope with accuracy as well as high throughput requirements. Surface tensions could be derived with a low sample consumption (800 μL) and a high reproducibility (<0.1‰ for water) within a reasonable time (3.5 min per sample). This method was used as a non-invasive HTP compatible approach to determine surface tensions of protein solutions dependent on protein content. The protein influence on the solutions' surface tension was correlated to the hydrophobicity of lysozyme, human lysozyme, BSA, and α-lactalbumin. Differences in proteins' hydrophobic character depending on pH and species could be resolved. Within this work we have developed a pH dependent hydrophobicity ranking, which was found to be in good agreement with literature. For the studied pH range of 3-9 lysozyme from chicken egg white was identified to be the most hydrophilic. α-lactalbumin at pH 3 exhibited the most pronounced hydrophobic character. The stalagmometric method occurred to outclass the widely used spectrophotometric method with bromophenol blue sodium salt as it gave reasonable results without restrictions on pH and protein species.