Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Design, expression and characterization of lactiscin—A novel broad-spectrum peptidic bacteriocin

Bacteria-derived antimicrobial peptides known as peptidic bacteriocins offer a promising alternative to traditional antibiotics in the face of the emergence of multidrug-resistant bacteria. Here, a nucleotide sequence of the gene encoding Lactococcus lactis-derived peptidic bacteriocin designated as lactiscin selectively identified from the GenBank® database was synthesized with an added 6⋅His sequence and cloned into Escherichia coli. Upon low-temperature expression at 16 °C, the His-tagged peptide could be produced in both soluble form and insoluble inclusions. Efficient purification of the soluble His-tagged peptide was achieved via immobilized-Ni2+ affinity chromatography (IMAC) and its estimated molecular mass of ∼13.4 kDa was determined by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified peptide was highly active against both Gram-positive and Gram-negative bacteria as it exhibited a minimal inhibitory concentration of 0.45 mg/mL, 0.15 mg/mL, 0.35 mg/mL and 0.45 mg/mL against. Escherichia coli, Vibrio parachemolyticus, Staphylococcus aureus and Micrococcus luteus, respectively. In addition, the lactiscin peptide still retained antimicrobial activity over a pH range of 3.0–12.0 and heat stability of 100 °C for 30 min. A membrane integrity study revealed that this peptidic bacteriocin was able to induce E. coli membrane permeabilization in a concentration-dependent manner, albeit it showed a negligible toxic effect on erythrocytic cells. Gel retardation assay demonstrated that the lactiscin bacteriocin could suppress the migration of genomic DNA extracted from pathogenic bacteria, suggesting the presence of bacteriocin-responsive binding genomic. Our findings of lactiscin—a novel broad-spectrum bacteriocin would be a valuable additive for the application of food industry as a potential bio-preservative.

Properties of exopolymeric substances (EPSs) produced during cyanobacterial growth: potential role in whiting events

Abstract. Extracellular polymeric substances (EPSs) are an important organic carbon reservoir in many pelagic and benthic environments. The production of EPS is intimately associated with the growth of phyto- and picoplankton. EPS plays a critical role in carbonate precipitation through the binding of cations and by acting as a nucleation site for minerals. Large-scale episodes of fine-grained calcium carbonate precipitation in the water column (whiting events) have been linked to cyanobacterial blooms, including of Synechococcus spp. The mechanisms that trigger these precipitation events are still debated. We pose that the cyanobacterial EPS, produced during exponential and stationary growth phases, plays a critical role in the formation of whitings. The aim of this study was to investigate the production of EPS during a 2-month cyanobacterial growth, mimicking a bloom. The production and characteristics of EPS were examined in different growth stages of Synechococcus spp. using various techniques such as Fourier transform infrared (FT-IR) spectroscopy as well as colorimetric and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) assays. We further evaluated the potential role of EPS in carbonate precipitation through in vitro-forced precipitation experiments. EPS produced during the early and late stationary phase contained a larger amount of negatively charged groups than present in EPS produced during the exponential phase. Consequently, a higher Ca2+-binding affinity of the stationary-phase EPS led to the formation of a larger amount of smaller carbonate minerals (< 50 µm) compared to crystals formed in exponential-phase EPS, which were less abundant and larger (> 50 µm). These findings were used to establish a conceptual model for picoplankton-bloom-mediated CaCO3 precipitation that can explain the role of EPS in whitings.

SDS-PAGE protein profile of albumin extracted by steaming from four marine and three brackish-water fishes

Abstract. Fatma N, Metusalach, Taslim NA, Nurilmala M. 2023. SDS-PAGE protein profile of albumin extracted by steaming from four marine and three brackish-water fishes. Biodiversitas 24: 4027-4033. Snakehead fish is a type of fish that has been known as the best source of fish albumin. However, the increasing demand for and exploitation of snakehead fish albumin is not accompanied by the availability of sustainable snakehead fish stocks. Snakehead fish stocks in nature continue to experience depletion and cultivation efforts that have been carried out so far have not yielded satisfactory results. For this reason, it is necessary to find alternative sources of fish albumin. The purpose of this study was to determine the molecular weight of the albumin proteins extracted by water bath steaming from four marine fishes (Indian scad Decapterus russelli, short-bodied mackerel Rastrelliger brachysoma, goldband fusilier Pterocaesio chrysozona, Japanese threadfin bream Nemipterus japonicus) and three brackish-water fishes (white-lipped eel catfish Paraplotosus albilabris, barramundi Lates calcarifer, milkfish Chanos chanos). The method used was Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis. Each sample was steamed in a water bath at 50°C for 30 minutes to obtain the crude extract's optimal yield and albumin content. The molecular weights of each protein band were analyzed using the Photocapt application, visualized and tabulated, and analyzed descriptively. Indian scad had six protein bands with three major (48 kD, 37 kD, 32 kD) and three minor bands (66 kD, 25 kD, 15 kD). Shortbodied mackerel had one major protein band (26 kD). Goldband fusilier had one major (33 kD) and four minor bands (50 kD, 69 kD, 57 kD, 26 kD). Japanese threadfin bream had one major (62 kD) and three minor bands (171 kD, 72 kD, 26 kD). White-lipped eel catfish had two major (41 kD, 34 kD and two minor bands (70 kD, 26 kD). Barramundi had one major (26 kD) and two minor bands (171 kD, 87 kD). Milkfish had one major (41 kD) and four minor bands (333 kD, 135 kD, 31 kD, 25 kD). It is hoped that this research will provide information on which fish have the potential as a source of fish albumin in terms of protein composition which resembles snakehead fish so that the properties provided are as good as albumin from snakehead fish.

Species recognition and the divergences in the chemical and ultrasonic signals between two coexisting Rattus species

Abstract The ability to recognize and differentiate between conspecifics and heterospecifics as well as their signals is critical for the coexistence of closely related species. In the genus Rattus, species are morphologically similar and multiple species often coexist. Here, we investigated the interspecific recognition and signal differentiation of two sympatric rat species, the brown rat (Rattus norvegicus, RN) and the Asian house rat (Rattus tanezumi, RT). In a two-way choice test, both RN and RT females showed a preference for conspecific male rats to heterospecific ones. RT females showed a significant preference for accessible urine of males of same species to those of other species, but not for the inaccessible urine. On the other hand, there were significant differences in the structural characteristics of the ultrasonic vocalization emitted by males of these two rat species. Sodium dodecyl sulphate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and isoelectric focusing electrophoresis unveiled that major urinary proteins (MUPs) in voided urine were more highly expressed in RN males versus RT males. The interspecific differences of urinary volatile compounds were also discussed. In conclusion, female rats had the ability to distinguish between males of either species.

Biotinylated caffeic acid covalent binding with myofibrillar proteins in alkaline conditions: Identification of protein-phenol adducts and alterations in protein properties.

In this study, the effects of covalent interactions between myofibrillar proteins (MP) and caffeic acid (CA) were investigated. Protein-phenol adducts were identified by biotinylated caffeic acid (BioC) used as a substitution of CA. The total sulfhydryls and free amines content were decreased (p<0.05). The α-helix structure of MP increased (p<0.05) and MP gel properties enhanced slightly at low dosages of CA (10 and 50μM), and both were impaired significantly (p<0.05) at high dosages of CA (250 and 1250μM). Two prominent adducts of myosin heavy chain (MHC)-BioC and Actin-BioC were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which gradually increased at low concentrations of BioC (10 and 50μM), and raised significantly at the concentration of 1250μM. According to the correlation analysis, MHC-BioC and Actin-BioC adducts showed a significant negative correlation with gel properties, such as G', hardness, and water holding capacity (WHC) (p<0.01), which indicated that the covalent interactions between MP and CA significantly affected the quality of meat products.

Cell clusters in intervertebral disc degeneration: an attempted repair mechanism aborted via apoptosis.

Cell clusters are a histological hallmark feature of intervertebral disc degeneration. Clusters arise from cell proliferation, are associated with replicative senescence, and remain metabolically, but their precise role in various stages of disc degeneration remain obscure. The aim of this study was therefore to investigate small, medium, and large size cell-clusters. For this purpose, human disc samples were collected from 55 subjects, aged 37-72 years, 21 patients had disc herniation, 10 had degenerated non-herniated discs, and 9 had degenerative scoliosis with spinal curvature <45°. 15 non-degenerated control discs were from cadavers. Clusters and matrix changes were investigated with histology, immunohistochemistry, and Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Data obtained were analyzed with spearman rank correlation and ANOVA. Results revealed, small and medium-sized clusters were positive for cell proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) in control and slightly degenerated human discs, while large cell clusters were typically more abundant in severely degenerated and herniated discs. Large clusters associated with matrix fissures, proteoglycan loss, matrix metalloproteinase-1 (MMP-1), and Caspase-3. Spatial association findings were reconfirmed with SDS-PAGE that showed presence to these target markers based on its molecular weight. Controls, slightly degenerated discs showed smaller clusters, less proteoglycan loss, MMP-1, and Caspase-3. In conclusion, cell clusters in the early stages of degeneration could be indicative of repair, however sustained loading increases large cell clusters especially around microscopic fissures that accelerates inflammatory catabolism and alters cellular metabolism, thus attempted repair process initiated by cell clusters fails and is aborted at least in part via apoptosis.

Structural insights into acidic heating-induced amyloid fibrils derived from soy protein as a function of protein concentration

Amyloid fibrillation of food-derived dietary proteins has been increasingly accepted as an effective approach to enhance protein functionality. Soy protein, an abundant and environmentally sustainable protein, has been widely used in food applications. However, the mechanism for amyloid fibrillation at various protein concentrations during the acidic heating process is not fully understood, along with an incomplete understanding of the structural polymorphism. In this study, we systematically investigated the influence of protein concentration on amyloid fibrillation of soy protein by means of atomic force microscopy (AFM), 1-anililo-naphthalene-8-sulfonate (ANS) and thioflavin T (Th T) fluorescence, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectrometer (FTIR). AFM analysis showed that soy protein-derived amyloid fibrils (SAFs) demonstrated very rich and diverse polymorphism and exhibited at least five kinds of long-rigid fibrils that had distinct periodicity, height, and handedness. The polypeptides hydrolysis behavior and the parallel to antiparallel β-sheet ratios were found to be protein concentration-dependent. Results obtained from this study will lead to a deeper understanding of formation mechanism of SAFs and lay the foundation for SAFs severing as functional ingredients in food field.

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)–derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

High‐performance enrichment of casein and ovalbumin in wall painting with immunomagnetic beads

AbstractThe detection of trace binding medium in wall paintings is usually challenging due to the aging and degradation of proteinaceous binding medium in a long time. Therefore, immunomagnetic beads (IMBs) with specific enrichment ability for casein and ovalbumin were prepared, and then the beads were applied for capturing target proteinaceous binding medium from ancient Chinese wall paintings. After optimization of experimental conditions, the enrichment performance of magnetic beads was confirmed by enzyme‐linked immunosorbent assay (ELISA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). In this experiment, archaeological wall painting samples were used to verify the accuracy of the construction enrichment method. The results showed that the OD450nm value of archaeological samples containing binding medium has increased to varying degrees after IMBs enrichment, effectively avoiding the occurrence of false negative results, indicating that the IMBs enrichment method developed in this work could be successfully applied for enriching ovalbumin and casein binding medium from ancient wall paintings for subsequently immunoassay detection.

Western Blotting with Fast SDS-PAGE and Semi-Dry Protein Transfer.

Western blotting is a method widely used in cell and molecular biology for the specific detection of proteins in biological samples. It is time-consuming and normally takes up to 2 days to complete. This protocol introduces a more time-efficient method to complete western blots, covering the preparation of protein extracts (including strategies for solubilization), electrophoretic separation of proteins, transfer of proteins to the membrane, and probing with antibodies. We describe an SDS-PAGE protocol that achieves a gradient-like separation of proteins (10-400 kDa) on a single-percentage polyacrylamide gel in only 45 min. Additionally, we present a rapid (10-14 min) semi-dry transfer of proteins from standard Tris/glycine polyacrylamide gels onto a membrane using homemade Tris/HEPES- or Tris/EPPS-based buffers. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Support Protocol 1: Cell lysis and protein extraction Support Protocol 2: Protein quantification with BCA assay and sample preparation for loading on gel Basic Protocol 2: Protein transfer with a fast semi-dry transfer (FSDT) buffer Basic Protocol 3: Immunoprobing, chemiluminescent visualization, stripping, and reuse of membranes.

Characterization of wheat flour millstreams for friabilin prevalence and nutrient composition

ABSTRACT In the current study, wheat varieties Inqulab-91 and AS-2002 were milled from a roller mill and analyzed for chemical characteristics through Near Infra-Red spectroscopy. Friabilin was extracted with Triton X-114 buffer and evaluated on sodium dodecyl sulfate polyacrylamide gel electrophoresis against known molecular weight protein standards. Current results show that the chemical constituents and friabilin revealed significant variation and uneven distribution among flour streams. In both wheat varieties, break streams showed higher protein, moisture, gluten, Zeleny and ash values. Inqualb-91 break streams had a higher lipid content than AS-2002 flour streams, but no clear trend was identified. The 15 kDa band was absent in M3, T2, sizing and low-grade flour of Inqulab-91 while the faint band was present in the remaining streams. Among the remaining AS-2002 mill streams, B2, B4, M3, M5, T1, and sizing showed a prominent 15 kDa band, while the rest showed a faint band. Friabilin depicted a positive correlation with all constituents except fat in AS-2002 while a negative correlation with gluten, Zeleny and fat contents for Inqulab-91.

Intestine-targeted high internal phase Pickering emulsion formulated using silkworm pupa protein via ultrasonic treatment

High internal phase Pickering emulsions (HIPPEs) stabilized by food grade particles have received much attention as deliver vehicles for bioactives in recent years. In this study, ultrasonic treatment was conducted to regulate the size of silkworm pupa protein (SPP) particles, fabricating oil–in–water (O/W) HIPPEs with intestinal releasability. Briefly, the pretreated SPP and SPP-stabilized HIPPEs were characterized, and the targeting release was investigated using in vitro gastrointestinal simulations and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Results revealed ultrasonic treatment time was the key factor regulating emulsification performance and stability of HIPPEs. Optimized SPP particles were obtained based on their size and zeta potential of 152.67 nm and 26.77 mV, respectively. With ultrasonic treatment, the hydrophobic groups in the secondary structure of SPP were exposed, facilitating the formation of a stable oil–water interface for HIPPEs. Additionally, SPP-stabilized HIPPE showed high stable against the gastric digestion. The SPP with 70 kDa molecular weight, which was the major interfacial proteins of the HIPPE, can be hydrolyzed by intestinal digestive enzymes, enabling the intestine-targeted release of the emulsion. Overall, in the present study, a facile method was developed to stabilize HIPPEs using solo SPP with ultrasonic treatment to protect and deliver hydrophobic bioactive ingredients.

Expression and characterization of recombinant IL-1Ra in Aspergillus oryzae as a system

BackgroundThe interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae.ResultsWe purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM).ConclusionThis study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.

Three-phase partitioning constructed by pH-responsive deep eutectic solvents and sugars for purification of radish (Raphanus sativus L.) peroxidase

Ammonium sulfate and t-butanol are widely used in conventional three-phase partitioning (TPP); however, their use is not eco-friendly. In this study, a novel TPP was developed using pH-responsive deep eutectic solvents (DESs) and sugars as substitutes for t-butanol and ammonium sulfate, respectively. This TPP was further used for the separation and purification of radish (Raphanus sativus L.) peroxidase (POD). DES-3 (dodecanoic acid: decanoic acid = 1:1) and sucrose were selected as optimal phase-forming components. Single-factor experiments were performed to investigate the factors influencing the TPP process, and three major factors (sucrose concentration, extraction time, and extraction temperature) were optimized by response surface methodology (RSM). Under the optimized conditions, the POD recovery and purification fold reached 101.66% and 11.56, respectively. Cyclic experiments confirmed the retrievability of DESs. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the novel TPP had better purification efficiency than the traditional TPP. The method proposed in this study is more efficient than conventional TPP, and it does not require the use of organic solvents and high concentration salt solutions. Therefore, this method is promising in the application of TPP.

Effect of gluconic acid rinsing on cadmium decontamination from rice protein.

Cadmium (Cd) accumulation in rice protein has long been considered a significant threat to human health. In the present study, a costless and effective method based on gluconic acid (GA) rinsing of rice protein was developed to reduce Cd contamination in rice protein. Moreover, the effect of GA on the structural and functional properties of rice protein was evaluated. With liquid-solid ratio of 30mL/g and oscillation time of 120min, 96.0% and 93.6% of Cd were eliminated from rice protein-H and rice protein-L, respectively. In addition, the results of scanning electron microscopy, Fourier transform infrared, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that GA treatment did not significantly change the structural properties of rice protein. However, GA treatment increased foaming properties, water holding capacity, and oil holding capacity of the rice protein, without affecting its further applicability. Thus, the proposed GA rinsing method can be considered a green and efficient strategy to solve the issue brought by Cd residual contamination in rice protein. PRACTICAL APPLICATION: Given the advantages of green and efficient agriculture, gluconic acid (GA) has emerged as a powerful strategy for removing the Cd from rice protein. The method developed herein showed great potentials for applications in the manufacture of rice-based products.

Insights into Intramuscular Connective Tissue Associated with Wooden Breast Myopathy in Fast-Growing Broiler Chickens.

Wooden breast myopathy (WBM) is a meat abnormality affecting pectoralis majors (PMs) of fast-growing broiler chickens. WBM-affected PMs exhibited varied meat qualities with increasing WBM severity. Normal PMs (NOR), mild WBM-affected PMs (MIL), moderate WBM-affected PMs (MOD), and severe WBM-affected PMs (SEV) were selected as raw materials. The structure and organization of connective tissue and fibrillar collagen were investigated through immersing with sodium hydroxide solution, Masson trichrome staining, and using an electron microscope. The mechanical strength of intramuscular connective tissue was analyzed via the shear force of samples treated with sodium hydroxide solution. The thermal property and secondary structure of connective tissue were analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The obtained connective tissue was dissolved in a sodium hydroxide solution for the evaluation of the physicochemical properties of proteins, including particle size, molecular weight, surface hydrophobicity, and intrinsic fluorescence. In particular, the particle size was measured using a zeta potential instrument. The molecular weight was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The surface hydrophobicity and intrinsic fluorescence were measured by spectroscopy technology. Histologically, macrophage infiltration, myodegeneration and necrosis, regeneration, fibrous connective tissue, and thickened perimysial connective tissue were observed in WBM-affected PMs, especially SEV with fibrosis, including blood vessels. Compared with NOR, WBM led to increased average diameter of the collagen fibrils in perimysial (36.61 nm of NOR to 69.73 nm of SEV) and endomysial (34.19 nm of NOR to 56.93 nm of SEV) layers. A significant increase (p < 0.05) was observed in the mechanical strength (2.05 N to 5.55 N) of fresh PMs and the thermal transition temperature (onset temperature (TO), 61.53 °C to 67.50 °C; maximum transition temperature (TM), 66.46 °C to 70.18 °C; termination temperature (TE), 77.20 °C to 80.88 °C) of connective tissue from NOR to SEV. Cooking decreased the mechanical strength, and MOD samples showed the highest mechanical strength (1.24 N, p < 0.05), followed by SEV (0.96 N), MIL (0.93 N), and NOR (0.72 N). For proteins in connective tissue, random coil (19.64% to 29.61%, p < 0.0001), particle size (p < 0.05), and surface hydrophobicity (p < 0.05) increased with the decrease in the α-helix (14.61% to 11.54%, p < 0.0001), β-sheet (45.71% to 32.80%, p < 0.0001), and intrinsic fluorescence of proteins from NOR to SEV. The molecular weights of intramuscular connective tissue proteins were in the ranges of >270 kDa, 180-270 kDa, 110-180 kDa, 95-100 kDa, and <15 kDa. Taken together, WBM resulted in thickened organization, tightly packed collagen fibrils, increased mechanical strength and thermal temperature, and increased particle size, surface hydrophobicity, and intrinsic fluorescence of proteins in connective tissue, as the WBM severity increased.

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Research on Repressing Allergen Cry j 1 Released from Japanese Cedar Pollen Using Todomatsu Oil

Japanese cedar (JC, Cryptomeria japonica) pollen allergens are the primary cause of JC pollinosis, a widespread seasonal allergic disorder and a significant public health issue in Japan. Nevertheless, rare information on repressing the pollen allergens released from JC pollen is available. This study aims to validate the repression of allergen Cry j 1 (the dominant JC pollen allergen that triggers JC pollinosis) using todomatsu oil produced from Abies sachalinensis waste, through surface plasmon resonance (SPR) experiments, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blind docking simulation. The findings revealed that todomatsu oil reduces the antibody reactivity and content of allergen Cry j 1 by 85.96% and 42.12%, respectively. The docking modeling suggested that alkyl hydrophobic forces, pi–sigma bonding, and hydrogen bonding are the principal motivating forces for todomatsu oil components to dock with allergen Cry j 1. More than 50% of the amino-acid residues docked by each todomatsu oil component (except α-pinene) are hydrophobic. Furthermore, oil components, especially β-maaliene, exhibited potent repression of allergen Cry j 1. Consequently, todomatsu oil, due to its inexpensive, available, convenient, and ecologically beneficial nature, is a viable measure to repress allergen Cry j 1.

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7‐501 using ion‐exchange chromatography, hydrophobic interaction chromatography, and Rotofor® isoelectric focusing

AbstractHyalophora cecropia pupae were infected by Enterobacter cloacae C7‐501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Ion‐exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non‐specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7‐501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.

Improvement of functional characteristics of Hypophthalmichthys molitrix protein by modification with chitosan oligosaccharide.

In the food processing field, it is very often that fish proteins are denatured affecting the nutritional value of the product which is vital to be solved. By using appropriate sugar donors for glycosylation with protein, improving the stability and emulsification properties of fish proteins can be achieved. This research looks into the impacts of enzymatic chitosan oligosaccharide (CO) at various concentration (0.15%, 0.30%, 0.45%, 0.60%, w/v) upon the molecular makeup and function of silver carp myofibrillar protein (MP) in an attempt to comprehend the impact of electrostatic binding among MP as well as CO on protein conformation. Analysis was done on the impact of various CO concentrations upon MP's secondary structure, conformational changes, and functional characteristics. Twelve sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assays were implemented to monitor MP; Fourier transform infrared spectroscopy, endogenous fluorescence spectroscopy, and UV absorption spectra were carried out to investigate the influence of CO on MP; Particle size distribution, emulsifying activity index (EAI), solubility, turbidity, sulfhydryl content, carbonyl content, foaming capacity, surface hydrophobicity, emulsifying stability index (ESI), and foam persistence were all investigated. In addition, we used dynamic light scattering, scanning electron microscope, and atomic force microscope to analyze myosin (MO) and 0.60% CO-MO complex. The results demonstrated that CO and MP form complexes through hydrogen bonding and electrostatic interactions. CO modification not only delayed the oxidation of MP but also promoted MP to show better solubility, foaming, and foaming stability. In addition, CO modified myosin particle size decreased, reducing myosin's roughness and making myosin's structure more compact. To sum up, molecular interaction could change functional characteristics, and products with special properties could be developed after modification with chitosan oligosaccharide.