Use Of Proteins Research Articles

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

The use of thermostable fluorescent proteins for live imaging in Sulfolobus acidocaldarius.

Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. We expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Of the tested proteins, thermal green protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. These initial results in live protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms.

Enhancing Functional Properties and Protein Structure of Almond Protein Isolate Using High-Power Ultrasound Treatment.

The suitability of a given protein for use in food products depends heavily on characteristics such as foaming capacity, emulsifiability, and solubility, all of which are affected by the protein structure. Notably, protein structure, and thus characteristics related to food applications, can be altered by treatment with high-power ultrasound (HUS). Almonds are a promising source of high-quality vegetable protein for food products, but their physicochemical and functional properties remain largely unexplored, limiting their current applications in foods. Here, we tested the use of HUS on almond protein isolate (API) to determine the effects of this treatment on API functional properties. Aqueous almond protein suspensions were sonicated at varying power levels (200, 400, or 600 W) for two durations (15 or 30 min). The molecular structure, protein microstructure, solubility, and emulsifying and foaming properties of the resulting samples were then measured. The results showed that HUS treatment did not break API covalent bonds, but there were notable changes in the secondary protein structure composition, with the treated proteins showing a decrease in α-helices and β-turns, and an increase in random coil structures as the result of protein unfolding. HUS treatment also increased the number of surface free sulfhydryl groups and decreased the intrinsic fluorescence intensity, indicating that the treatment also led to alterations in the tertiary protein structures. The particle size in aqueous suspensions was decreased in treated samples, indicating that HUS caused the dissociation of API aggregates. Finally, treated samples showed increased water solubility, emulsifying activity, emulsifying stability, foaming capacity, and foaming stability. This study demonstrated that HUS altered key physicochemical characteristics of API, improving critical functional properties including solubility and foaming and emulsifying capacities. This study also validated HUS as a safe and environmentally responsible tool for enhancing desirable functional characteristics of almond proteins, promoting their use in the food industry as a high-quality plant-based protein.

Silk fibroin wetting stability film induced by polyamide-amine-epichlorohydrin (PAE) for intelligent sensing system

Protein materials gain new functions and applicability through redesigns in protein structure and engineering confer. However, the application and development of proteins for use in flexible devices that fit in flexible devices that fit the surface of human skin is hindered by their poor wet stability. Here, we described the design of wet-stable materials based on the reconstruction of silk fibroin (SF). The combination of polyamide-amine-epichlorohydrin (PAE) was used as a traction rope to bring SF molecular chains closer to each other, to facilitate the self-assembly of SF through branching and lengthening of molecular chains, and change its crystalline structure. SF/PAE composite films that exhibited huge improvement in ductility and wet stability were combined with flexible SF substrates via patterning and ion sputtering to prepare flexible sensors. In addition, the SF/PAE sensing system equipped with a microprocessor and Bluetooth module enabled the real-time remote acquisition of human health signals such as vocal cords, joints, pulse and meridians. This reconfiguration of the SF structure will advance the systematic exploration of protein structures and the development of protein materials for intelligent device applications.

A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence.

The amyloid-beta precursor protein is a transmembrane protein expressed in many tissues and highly concentrated in the brain. The protein is of significant interest due to its involvement in the generation of amyloidogenic β-amyloid peptides, prone to plaque formation that is characteristic of Alzheimer's Disease. The scientific community would benefit from the availability of high-quality anti-amyloid-beta precursor protein antibodies to enhance reproducible research on this target. In this study, we characterized eleven amyloid-beta precursor protein commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Antibacterial Profiling of Zanthoxylum armatum Extracts: A Comprehensive Computational and Experimental Study

Objectives The current study was conducted to evaluate the antibacterial potential of leaf and fruit extracts of Zanthoxylum armatum against two pathogenic bacterial isolates, Staphylococcus aureus and Staphylococcus epidermidis. Methods Twelve commercially available antibiotics were tested S. aureus and S. epidermidis by antimicrobial susceptibility test (AST). Qualitative analysis of phytochemicals was performed to evaluate the presence of certain secondary metabolites. The activity of Z. armatum extracts against S. aureus and S. epidermidis was measured as a maximum zone of inhibition exhibited by each leaf and fruit extract. An in-silico study was conducted on flavonoids and alkaloids to show their binding affinity with the PBP2a receptor protein of S. aureus and TcaR of S. epidermidis. Results The AST revealed that S. aureus was resistant to Penicillin, Ampicillin, Clindamycin, Vancomycin, Rifampicin, Novobiocin, and Oxacillin, whereas S. epidermidis was resistant to Streptomycin, Oxacillin, Tetracycline, and Novobiocin. Qualitative analysis of phytochemicals resulted in the presence of Saponins, fixed oils, flavonoids, alkaloids, starch, and fatty acids in both leaf and fruit extracts. The maximum zone of inhibition against S. aureus was produced by methanolic leaf extracts of Z. armatum and chloroform fruit extracts. For S. epidermidis, the best activity was exhibited by benzene leaf extracts and methanolic fruit extracts. An in-silico study showed that flavonoids Nitidine and Nevadensin exhibited binding affinity with the PBP2a receptor protein higher than selected antibiotics, ie, Penicillin, Chloramphenicol, and Oxacillin. TcaR of S. epidermidis interacted with Tambuletin, followed by Nitidine and Kaempferol. Conclusion After in vitro testing, in silico analysis advised extracting and purifying the bioactive components from Z. armatum extracts that showed significant interaction with bacterial virulence proteins for use as natural antibiotics against antibiotic-resistant bacteria.

Biophysical and biochemical characterization of a recombinant Lyme disease vaccine antigen, CspZ-YA

Lyme disease, caused by Lyme Borrelia spirochetes, is the most common vector-borne illness in the United States. Despite its global significance, with an estimated 14.5 % seroprevalence, there is currently no licensed vaccine. Previously, we demonstrated that CspZ-YA protein conferred protection against Lyme Borrelia infection, making it a promising vaccine candidate. However, such a protein was tagged with hexahistidine, and thus not preferred for vaccine development; furthermore, the formulation to stabilize the protein was understudied. In this work, we developed a two-step purification process for tag-free E. coli-expressed recombinant CspZ-YA. We further utilized various bioassays to analyze the protein and determine the suitable buffer system for long-term storage and formulation as a vaccine immunogen. The results indicated that a buffer with a pH between 6.5 and 8.5 stabilized CspZ-YA by reducing its surface hydrophobicity and colloidal interactions. Additionally, low pH values induced a change in local spatial conformation and resulted in a decrease in α-helix content. Lastly, an optimal salinity of 22–400 mM at pH 7.5 was found to be important for its stability. Collectively, this study provides a fundamental biochemical and biophysical understanding and insights into the ideal stabilizing conditions to produce CspZ-YA recombinant protein for use in vaccine formulation and development.

Thermal characterization and separation of whey proteins by differential scanning calorimetry

Most commercially available whey products contain a mixture of 6–7 whey proteins; however, there is an increased focus on using the individual whey proteins for their unique biological activities. Before extracting individual whey proteins for use, it is important to quantify how much of a particular protein is present in whey mixtures as well as if the protein is still structurally folded. We first characterized the denaturation temperature and enthalpy values for the six purified whey proteins at six pHs (3–9) and under ion chelation using a nano-differential scanning calorimeter (DSC). From the individual protein scans, we determined the optimal condition for detecting all 6 proteins on a single DSC scan was whey in an EDTA MOPs pH 6.7 buffer.

Combining colloid milling and twin screw pressing for oleosome extraction

Oleosomes are oil droplets abundant in oilseeds that can be extracted together with proteins for use in emulsion-like foods and can be used as natural, ready emulsions as alternatives to dairy and fat-based products. To make optimal use of the raw material, we look at simultaneous extraction of oleosomes and proteins from rapeseed. The combined approach of colloid milling and twin screw pressing resulted in a higher yield of oleosomes (70 % w/w) when compared to twin screw press alone (54 % w/w), which may be due to extensive cell wall disruption caused by colloid mill processing, as evidenced by SEM imaging. The protein yield remained constant irrespective of the degree of cell wall disruption (50 % w/w). In conclusion, combining the colloid mill and twin screw press is a straightforward way for the extraction of oleosome and proteins from rapeseeds for food application.

Effect of Ultrasonic-Assisted Enzymatic Hydrolysis on Functional Properties and Antioxidant Activity of Eri Silkworm Pupa Protein Isolate

Philosamia ricini (Eri silkworm) pupa protein isolate (EPI) was utilized to prepare pupa protein hydrolysate (EPIH) through enzymatic hydrolysis. Additionally, the isolate underwent ultrasonic treatment at 20 kHz to become ultrasound pretreated EPI (EPIU), which was then enzymatically hydrolyzed to obtain ultrasound pretreated protein hydrolysate (EPIUH). The physicochemical properties of these samples were investigated, including molecular weight, solubility, foaming and emulsion properties, water- and oil-holding capacity, antioxidant activity, and color. When compared to EPI (used as the control), EPIU exhibited a high degree of hydrolysis at 20 minutes ( DH = 29.24 % ). At a total process time of 20 minutes, the degree of hydrolysis for EPIH, EPIU, and EPIUH was found to be 13%, 29%, and 41%, respectively. SDS-PAGE analysis indicated no difference in molecular weight between EPI and EPIU (11–75 kDa). However, the molecular weight profiles of EPIH and EPIUH were reduced (8–45 kDa), resulting in changes in protein functionalities. The high DH value contributed to the enhancement of antioxidant activity, solubility, emulsion capacity, emulsion stability, and foam capacity of the protein isolate at pH 7. Furthermore, the ultrasonic pretreatment of the protein hydrolysate increased the lightness of the protein powder by reducing the enzyme activity of the polyphenol oxidase (PPO). These results suggest that ultrasonic pretreatment of the protein hydrolysate could be applied to improve the properties of Eri silkworm pupa protein for use in the food and beverage industry, such as protein-rich beverages or salad dressings.

Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence

The amyloid-beta precursor protein is a transmembrane protein expressed in many tissues and highly concentrated in the brain. The protein is of significant interest due to its involvement in the generation of amyloidogenic β-amyloid peptides, prone to plaque formation that is characteristic of Alzheimer’s Disease. The scientific community would benefit from the availability of high-quality anti-amyloid-beta precursor protein antibodies to enhance reproducible research on this target. In this study, we characterized eleven amyloid-beta precursor protein commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

A new ITO-based Aβ42 biosensor for early detection of Alzheimer's disease

In this study, a novel label-free impedimetric immunosensor was fabricated for rapid, selective, and sensitive quantitative analysis of Aβ42 protein for use in the diagnosis of Alzheimer's disease. The immunosensor was fabricated using inexpensive and disposable indium tin oxide polyethylene terephthalate (ITO-PET) electrodes. After the electrodes were modified with 3-glycidoxypropyldimethoxymethylsilane (GPDMMS), the antibody specific to the Aβ42 protein (anti-Aβ42) was immobilized. The affinity interaction between anti-Aβ42 and Aβ42 in the immobilization steps in immunosensor fabrication and in the quantitation of Aβ42 were analyzed using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) techniques. Additionally, the morphological changes occurring on the electrode surface during each immobilization step were imaged using scanning electron microscopy (SEM). The linear detection range of the immunosensor was determined as 1–100 pg/mL with the limit of detection value of 0.37 pg/mL. Analytical properties of the biosensor, including reproducibility, repeatability, storage stability, selectivity, and regeneration were investigated. The kinetic behavior of antibody-antigen complex formation was determined for the first time using single frequency impedance (SFI) analysis on an Aβ42 biosensor. The potential for use of the immunosensor in clinical studies was demonstrated by analysis of Aβ42 in commercially purchased human serum.

Life Cycle Assessment of Black Soldier Fly, Yellow Mealworm and Soybean Protein for Use in Fish Feed

As the consumption of fish in the human diet increases, a larger amount of production is needed. The growing demand for fish also has an impact on fish feed, its production efficiency and the sustainability of using raw materials. To evaluate the sustainability of raw materials and the impact on the environment, three protein alternatives are compared – black soldier fly, yellow mealworm and soybean. Each alternative has advantages and disadvantages. The advantages of black soldier fly and yellow mealworm are a valuable source of protein, sustainable growth (as feed can be used for food waste) and no need for arable land. Disadvantages of black soldier fly and yellow mealworm are price, an unbalanced diet can negatively affect growth, and nutritional value effect on the fish vary depending on fly or mealworm species. The advantages of soybeans are price, availability and high protein content, but the disadvantages are a lack of essential amino acids that affect the quality of fish and poor palatability. An LCA study has been carried out for the black soldier fly, yellow mealworm and soybean protein. From PEFCR most relevant impact categories are climate change, particulate matter, acidification, land use, eutrophication terrestrial and water use. The total single score value for black soldier fly protein is 1.43E+01 μPt, for yellow mealworm protein is 3.89E+02 μPt and for soybean protein is 9.72E+01 μPt. Large impact is from electricity consumption, used components for feed. Sensitivity analysis was performed for black soldier fly protein and yellow mealworm protein production, where feed composition was changed. In sensitivity analysis environmental impact is less from the new feed structure. The reason might be that the new feed structure has used food waste and wheat as feed ingredients.

New Aequorea Fluorescent Proteins for Cell-Free Bioengineering.

Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms.

Engineering thermostable affinity proteins for use in high-throughput immunoassay formats

Protein engineering approach enables effective immobilisation of thermostable affinity proteins onto polystyrene materials.

Effect of Limited Enzymatic Hydrolysis on Structural and Functional Properties of Elaeagnus mollis Oil Meal Protein.

Elaeagnus mollis oil (EMO) meal, a by-product of oil production with plentiful protein, is considered a cheap and good quality source of plant protein for use in the food industry. In this study, the influence of limited enzymatic hydrolysis of EMO meal protein on the structure, solubility, foaming and emulsifying capacities was investigated in detail. The hydrolysates with different DH values (5, 10, 15, and 17) were obtained by controlling the time of enzymatic hydrolysis with alcalase. The results showed that enzymatic hydrolysis decreased molecular weight and increased flexibility and surface hydrophobicity. At the given range of pH and concentration of NaCl, the solubility, foaming and emulsifying capacities of hydrolysates were significantly improved, especially in the area of isoelectric point, and increased with the increase of DH. It was also found that the hydrolysate with DH10 had better foaming and emulsifying stability. In general, appropriate enzymatic hydrolysis could improve the functional properties in favor of their potential use as food ingredients.

Effects of Pea Protein on Satiety, Postprandial Glucose Response and Appetite Hormones: A Literature Review

Introduction: Type 2 Diabetes (T2D) is one of the leading causes of mortality with obesity being one of the greatest risk factors. Increased protein intake has been found to increase satiety, that could potentially aid in weight control. However, much of the research is elusive on the specifics of the effects of plant-based protein, specifically pea protein on satiety and responses linked to appetite. The purpose of this review was to investigate the effects of pea protein on satiety, postprandial glucose response and appetite. Methods: Studies of the existing literature were found, filtered, and analyzed from scientific databases Cochrane Library, PubMed, ScienceDirect, and Web of Science entering a combination of the keywords “pea protein”, “satiety”, and “postprandial response”. A total of 11 articles were analyzed to determine the relationship between pea protein consumption and postprandial response of satiety and appetite. Results: Pea protein consumption as a preload increased satiety and lowered food intake between 30 and 120 minutes after ingestion. Postprandial blood glucose was lowered and various appetite hormones increased at different time lapses. Discussion: Although the oral consumption of pea protein alone was seen to effectively induce satiety, other factors such as the addition of fibre, the method of administration, or rates of gastric emptying could significantly affect food intake. Conclusion: This literature review establishes a link between plant proteins and its benefits of feelings of satiety and appetite to promote incorporating more plant proteins in the diet. Future research should further investigate the link between postprandial responses and appetite hormones to identify benefits of pea protein for use in the food industry and increase public consumption of pea protein.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae)

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407bp, while its mRNA sequence was recognized as 2115bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Immunostimulant plant proteins: Potential candidates as vaccine adjuvants.

The COVID‐19 pandemic is shaking up global scientific structures toward addressing antibiotic resistance threats and indicates an urgent need to develop more cost‐effective vaccines. Vaccine adjuvants play a crucial role in boosting immunogenicity and improving vaccine efficacy. The toxicity and adversity of most adjuvant formulations are the major human immunization problems, especially in routine pediatric and immunocompromised patients. The present review focused on preclinical studies of immunoadjuvant plant proteins in use with antiparasitic, antifungal, and antiviral vaccines. Moreover, this report outlines the current perspective of immunostimulant plant protein candidates that can be used by researchers in developing new generations of vaccine‐adjuvants. Future clinical studies are required to substantiate the plant proteins' safety and applicability as a vaccine adjuvant in pharmaceutical manufacturing.

Evaluation of four novel protein sources as alternatives to soybean meal for two specifications of cage-farmed grass carp (Ctenopharyngodon idellus) deeds: Effect on growth performance, flesh quality, and expressions of muscle-related genes

Costs associated with the use of soybean as a food source in aquaculture have imposed constraints on its use in this industry. Thus, research is now focusing on alternative sources of protein for use in aquaculture. Here, we assessed the effect of total replacement of soybean meal (SBM) by Clostridium autoethanogenum protein (CAP), Tenebrio molitor meal (TMM), cottonseed protein concentrate (CPC), and chlorella powder (CHP) in diets for small specimens (initial body weight: 239.72 ± 10.75 g) and large specimens (initial body weight: 638.32 ± 12.64 g) of grass carp. For that purpose, five isonitrogenous (300 g/kg) and isoaliphatic (58 g/kg) diets were formulated with SBM, CAP, TMM, CPC, and CHP as the only dietary protein source. Triplicate groups of fish were fed the experimental diets for 56 days. Results showed that irrespective of protein sources, large specimens of grass carp obtained the significantly reduced weight gain rate (WGR), specific growth rate, moisture, crude ash contents, drip loss rate, and gene expressions of muscle S6K, MyoD, and Myf-5, and the significantly increased crude protein and crude lipid contents, hardness, cohesiveness, adhesiveness, springiness, chewiness and shear force of muscles, and gene expressions of muscle TOR, 4EBP1, MyoG, MRF4, and MSTN-1. Regardless of size, the CHP group possessed markedly higher WGR, protein efficiency ratio, crude protein contents, hardness, adhesiveness, chewiness values, and gene expression levels of muscle TOR, 4EBP1, Myf-5, and MRF4, and significantly lower feed conversion rate, drip loss rate, and gene expressions of muscle S6K and MSTN-1. For small specimens or for large specimens, the essential amino acid contents of the CHP group were highest, and CHP markedly elevated the delicious amino acid and glycine contents in comparison with the SBM. Moreover, the CPC group also realized the function of promoting the growth and improving the flesh quality to a certain degree. However, total replacement of soybean meal by CAP and TMM in grass carp diets restrained the growth and damaged the flesh quality in varying degrees. Overall, comparatively, total replacement of SBM with CHP not only improved growth performance and feed use, but also enhanced the flavor and texture of the fish by improving the amino acid composition and water-holding capacity of muscle, as well as improving muscle growth by positively impacting the expression of genes associated with the regulation of growth and development. Therefore, CHP could be a potential protein source to replace SBM in diets for use in the aquaculture of grass carp.