Establishing a novel ternary complex of soybean protein isolated-tannic acid-magnesium ion and its properties.

A ternary complex composed of soybean protein isolated (SPI), tannic acid (TA) and magnesium ion (M) was established to enhance the capability of protein carriers for TA delivery. SPI was firstly covalently bind with TA (TA-SPI) and then M was employed to form the ternary complex (M-TA-SPI). Their structures, gel and digestion properties were further investigated. TA was observed to covalently bind with SPI. TA-SPI and M-TA-SPI complexes showed different molecule size and spatial structures after binding with M and TA. The increasing of TA amount changed the intramolecular interactions, microstructure and texture properties of M-TA-SPI gels. Compared with TA-SPI, M retarded the gastric digestion of M-TA-SPI and caused higher TA release amount in intestinal tract. In this study, M-TA-SPI was determined to be a good carrier to protect and release TA in gastrointestinal digestion. This kind of complex may have potential applications for loading polyphenols in nutraceuticals.

Hydroxyapatite-based composites: Excellent materials for environmental remediation and biomedical applications.

Given their unique characteristics and properties, Hydroxyapatite (HAp) nanomaterials and nanocomposites have been used in diverse advanced catalytic technologies and in the field of biomedicine, such as drug and protein carriers. This paper examines the structure and properties of the manufactured HAp as well as a variety of synthesis methods, including hydrothermal, microwave-assisted, co-precipitation, sol-gel, and solid-state approaches. Additionally, the benefits and drawbacks of various synthesis techniques and ways to get around them to spur more research are also covered. This literature discusses the various applications, including photocatalytic degradation, adsorptions, and protein and drug carriers. The photocatalytic activity is mainly focused on single-phase, doped-phase, and multi-phase HAp, while the adsorption of dyes, heavy metals, and emerging pollutants by HAp are discussed in the manuscript. Furthermore, the use of HAp in treating bone disorders, drug carriers, and protein carriers is also conferred. In light of this, the development of HAp-based nanocomposites will inspire the next generation of chemists to improve upon and create stable nanoparticles and nanocomposites capable of successfully addressing major environmental concerns. This overview's conclusion offers potential directions for future study into HAp synthesis and its numerous applications.

Enriching alginate matrix used for probiotic encapsulation with whey protein concentrate or its trypsin-derived hydrolysate: Impact on antioxidant capacity and stability of fermented whey-based beverages.

The present research is part of an effort to create whey-based functional food. Previously, it was concluded that proteins and peptides in an encapsulation matrix contribute to mechanical properties of beads, fermentative activity, acid and bile tolerance, and the survival of probiotics during the simulated gastrointestinal condition. This research evaluates the effects of using whey protein concentrate and trypsin hydrolysate as components of a matrix for probiotic encapsulation on the antioxidant capacity of a beverage. Carrier with hydrolysate showed better encapsulation efficiency, spherical factor, and antioxidant capacity before and after fermentation compared to the carrier with non-hydrolyzed proteins. Hydrolysis of protein used for carrier formulation positively impacts the beverage's antioxidant properties and probiotic viability during 28 days of storage. Using proteins, especially peptides, as a matrix component achieved three objectives: protection of probiotics, enrichment of products with antioxidants, and neutralization of possible bitter taste (because the bitter tasting peptides are incorporated into the matrix and as such cannot contribute to the taste of the product) that bioactive peptides usually possess.

Quantitative Consequences of Protein Carriers in Immunopeptidomics and Tyrosine Phosphorylation MS2 Analyses.

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20× protein carrier in pMHC analyses and a high (~100×) and low (~9×) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the "no-boost" condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

pH-Sensitive Chitosan Nanoparticles for Salivary Protein Delivery.

Salivary proteins such as histatins (HTNs) have demonstrated critical biological functions directly related to tooth homeostasis and prevention of dental caries. However, HTNs are susceptible to the high proteolytic activities in the oral environment. Therefore, pH-sensitive chitosan nanoparticles (CNs) have been proposed as potential carriers to protect proteins from enzymatic degradation at physiological salivary pH. Four different types of chitosan polymers were investigated and the optimal formulation had good batch to batch reproducibility, with an average hydrodynamic diameter of 144 ± 6 nm, a polydispersity index of 0.15 ± 0.04, and a zeta potential of 18 ± 4 mV at a final pH of 6.3. HTN3 encapsulation and release profiles were characterized by cationic polyacrylamide gel electrophoresis. The CNs successfully encapsulated HTN3 and selectively swelled at acidic pH to facilitate HTN3 release. Protection of HTN3 against enzymatic degradation was investigated in diluted whole saliva. HTN3 encapsulated in the CNs had a prolonged survival time compared to the free HTN3. CNs with and without HTN3 also successfully reduced biofilm weight and bacterial viability. The results of this study have demonstrated the suitability of CNs as potential protein carriers for oral applications, especially for complications occurring at acidic conditions.

Proteins and Peptides as Important Modifiers of the Polymer Scaffolds for Tissue Engineering Applications-A Review.

Polymer scaffolds constitute a very interesting strategy for tissue engineering. Even though they are generally non-toxic, in some cases, they may not provide suitable support for cell adhesion, proliferation, and differentiation, which decelerates tissue regeneration. To improve biological properties, scaffolds are frequently enriched with bioactive molecules, inter alia extracellular matrix proteins, adhesive peptides, growth factors, hormones, and cytokines. Although there are many papers describing synthesis and properties of polymer scaffolds enriched with proteins or peptides, few reviews comprehensively summarize these bioactive molecules. Thus, this review presents the current knowledge about the most important proteins and peptides used for modification of polymer scaffolds for tissue engineering. This paper also describes the influence of addition of proteins and peptides on physicochemical, mechanical, and biological properties of polymer scaffolds. Moreover, this article sums up the major applications of some biodegradable natural and synthetic polymer scaffolds modified with proteins and peptides, which have been developed within the past five years.

Seasonal Weather Changes Affect the Yield and Quality of Recombinant Proteins Produced in Transgenic Tobacco Plants in a Greenhouse Setting.

Transgenic plants have the potential to produce recombinant proteins on an agricultural scale, with yields of several tons per year. The cost-effectiveness of transgenic plants increases if simple cultivation facilities such as greenhouses can be used for production. In such a setting, we expressed a novel affinity ligand based on the fluorescent protein DsRed, which we used as a carrier for the linear epitope ELDKWA from the HIV-neutralizing antibody 2F5. The DsRed-2F5-epitope (DFE) fusion protein was produced in 12 consecutive batches of transgenic tobacco (Nicotiana tabacum) plants over the course of 2 years and was purified using a combination of blanching and immobilized metal-ion affinity chromatography (IMAC). The average purity after IMAC was 57 ± 26% (n = 24) in terms of total soluble protein, but the average yield of pure DFE (12 mg kg-1) showed substantial variation (± 97 mg kg-1, n = 24) which correlated with seasonal changes. Specifically, we found that temperature peaks (>28°C) and intense illuminance (>45 klx h-1) were associated with lower DFE yields after purification, reflecting the loss of the epitope-containing C-terminus in up to 90% of the product. Whereas the weather factors were of limited use to predict product yields of individual harvests conducted for each batch (spaced by 1 week), the average batch yields were well approximated by simple linear regression models using two independent variables for prediction (illuminance and plant age). Interestingly, accumulation levels determined by fluorescence analysis were not affected by weather conditions but positively correlated with plant age, suggesting that the product was still expressed at high levels, but the extreme conditions affected its stability, albeit still preserving the fluorophore function. The efficient production of intact recombinant proteins in plants may therefore require adequate climate control and shading in greenhouses or even cultivation in fully controlled indoor farms.

Ion-exchanging dialysis as an effective method for protein entrapment in curdlan hydrogel.

The purpose of this study was to determine whether dialysis method allows for efficient protein entrapment in curdlan-based hydrogel. Thus, bovine serum albumin, a model of bioactive protein, was incorporated into curdlan matrix using ion-exchanging dialysis method against two concentrations of CaCl2 solution - 2% and 10%, respectively. Then, physicochemical, mechanical, and biological properties of the bovine serum albumin-loaded curdlan hydrogels were evaluated. Received results show that neither the polymer nor the entrapment procedure change the bovine serum albumin conformation (as proven by Fourier transform infrared spectroscopy and circular dichroism spectroscopy) and the process guarantees high protein entrapment efficiency (above 95%). The curdlan-based carrier obtained against 2% of CaCl2 solution was found to possess higher swelling ability, release greater amounts of bovine serum albumin (up to 4 weeks), and exhibit superior biocompatibility compared to curdlan-based carrier obtained against 10% of CaCl2 solution. Thus, dialysis method enables efficient protein entrapment in curdlan hydrogel and obtained protein carrier via dialysis method into 2% of CaCl2 solution may be considered as a promising protein delivery system especially for tissue engineering applications. It should be noted that we are the first who presented effective method for protein entrapment in curdlan hydrogel.

The Effect of Size, Dose, and Administration Route on Zein Nanoparticle Immunogenicity in BALB/c Mice.

BACKGROUND: Zein-based carriers are a promising delivery system for biomedical applications. However, few studies involve systematic investigation on their in vivo biocompatibility and immunogenicity. PURPOSE: The objective of this study was to identify the immunogenicity, type of immune response, biocompatibility and systemic recall immune response of zein nanoparticles administrated via different routes in mice. ANIMALS AND METHODS: Female Balb/c mice were selected as the animal model in this paper. The effect of particle size, dose and inoculation routes on immunogenicity were systematically explored. The mice were challenged at week 50 via intramuscular and subcutaneous routes to investigate the systemic recall immune responses of zein nanoparticles. Hematoxylin and eosin staining was performed to investigate the biocompatibility of zein nanoparticles at injection sites. RESULTS: The administration of zein particles by parenteral routes led to a long-term systemic immune response. Particle size did not affect zein-specific IgG antibody titers. IgG antibody titers and inflammatory cell infiltration at the injection sites resulting from intramuscular zein particle injection were significantly higher than those from subcutaneous injection of the same dose. For intramuscular inoculation, dose-dependent IgG antibody titers were observed after the third inoculation, while no significant difference was found via the subcutaneous route. For both routes, IgG titer showed a time-dependent decrease at all dose levels from week 5 onward, and finally plateaued at week 28. The IgG subtype assay showed a predominant Th2-type immune response for both administration routes. Challenge with zein nanoparticles at week 50 led to a significant increase in specific IgG titer at all dose levels, indicating systemic recall immune responses. Interestingly, IgG antibody levels in the subcutaneous groups showed a delayed decrease compared to those of the intramuscular injection groups at all dose levels. CONCLUSION: This study indicated that immunogenicity may be one of the key challenges of using zein nanoparticles as carriers via parenteral administration. Further investigation is needed to illustrate zein immunogenicity in other forms, and the possible effect of systemic recall immune response on in vivo pharmacokinetic characteristics.

A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies.

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity. Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution. We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

Protein delivery nanosystem of six-arm copolymer poly(ε-caprolactone)-poly(ethylene glycol) for long-term sustained release.

BACKGROUND: To address the issue of delivery of proteins, a six-arm copolymer, six-arm poly (ε-caprolactone)-poly(ethylene glycol) (6S-PCL-PEG), was synthesized by a simple two-step reaction. Thereafter, the application of 6S-PCL-PEG as a protein carrier was evaluated. MATERIALS AND METHODS: A six-arm copolymer, six-arm poly(ε-caprolactone) (6S-PCL), was synthesized by ring-opening polymerization, with stannous octoate as a catalyst and inositol as an initiator. Then, poly(ethylene glycol) (PEG) was linked with 6S-PCL by oxalyl chloride to obtain 6S-PCL-PEG. Hydrogen-1 nuclear magnetic resonance spectrum, Fourier-transform infrared spectroscopy, and gel-permeation chromatography were conducted to identify the structure of 6S-PCL-PEG. The biocompatibility of the 6S-PCL-PEG was evaluated by a cell counting kit-8 assay. Polymeric nanoparticles (NPs) were prepared by a water-in-oil-in-water double emulsion (W1/O/W2) solvent evaporation method. The size distribution and zeta potential of NPs were determined by dynamic light scattering. Transmission electron microscopy was used to observe the morphology of NPs. Drug-loading capacity, encapsulation efficiency, and the release behavior of ovalbumin (OVA)-loading NPs were tested by the bicinchoninic acid assay kit. The stability and activity of OVA released from NPs were detected and the uptake of NPs was evaluated by NIH-3T3 cells. RESULTS: All results indicated the successful synthesis of amphiphilic copolymer 6S-PCL-PEG, which possessed excellent biocompatibility and could formulate NPs easily. High drug-loading capacity and encapsulation efficiency of protein NPs were observed. In vitro, OVA was released slowly and the bioactivity of OVA was maintained for over 28 days. CONCLUSION: 6S-PCL-PEG NPs prepared in this study show promising potential for use as a protein carrier.

Alphavirus capsid proteins self-assemble into core-like particles in insect cells: A promising platform for nanoparticle vaccine development.

The mosquito-borne chikungunya virus (CHIKV) causes arthritic diseases in humans, whereas the aquatic salmonid alphavirus (SAV) is associated with high mortality in aquaculture of salmon and trout. Using modern biotechnological approaches, promising vaccine candidates based upon highly immunogenic, enveloped virus-like particles (eVLPs) have been developed. However, the eVLP structure (core, lipid membrane, surface glycoproteins) is more complex than that of non-enveloped, protein-only VLPs, which are structurally and morphologically 'simple'. In order to develop an alternative to alphavirus eVLPs, in this paper we engineered recombinant baculovirus vectors to produce high levels of alphavirus core-like particles (CLPs) in insect cells by expression of the CHIKV and SAV capsid proteins. The CLPs localize in dense nuclear bodies within the infected cell nucleus and are purified through a rapid and scalable protocol involving cell lysis, sonication and low-speed centrifugation steps. Furthermore, an immunogenic epitope from the alphavirus E2 glycoprotein can be successfully fused to the N-terminus of the capsid protein without disrupting the CLP self-assembling properties. We propose that immunogenic epitope-tagged alphavirus CLPs produced in insect cells present a simple and perhaps more stable alternative to alphavirus eVLPs.

Effect of net surface charge on physical properties of the cellulose nanoparticles and their efficacy for oral protein delivery.

Both net positively and negatively charged cellulose-based nanoparticles were prepared from oppositely charged carboxymethylcellulose (CMC) and quaternized cellulose (QC). Effect of surface charge on efficacy of cellulose nanoparticles for delivering both positively and negatively charged proteins was investigated. Lysozyme (LYS) and bovine serum albumin (BSA), which possess positive and negative charge at physiological pH respectively, were used as models. The results revealed that high encapsulation efficiency (67.7% and 85.1%) could be achieved when negatively charged protein was encapsulated in positively charged nanoparticles, or positively charged protein was encapsulated in negatively charged nanoparticles. Proteins encapsulated in optimal cellulose nanoparticles could be sustainably released and no obvious protein denaturation was detected. Both net positively and negatively charged nanoparticles exhibited low cytotoxicity due to cellulose's good biocompatibility. Not only net positively charged nanoparticles demonstrated high cellular uptake efficiency, but also net negatively charged nanoparticles showed somewhat efficient cellular uptake.

The effect of meningococcal serogroup A polysaccharide-protein conjugate vaccine prepared with TT and rCTB on mucosal immune responses / 中华微生物学和免疫学杂志

Objective To analyze the feasibility of the recombinant cholera toxin B subunit (rCTB) as a carrier protein candidate for the preparing of polysaccharide-protein conjugate, and to discuss the immune effects of tetanus toxoid (TT) as the carrier protein in mucosal delivery vaccine. Methods The refolded pentrumer protein, rCTB was obtained by genetic engineering methods. Then conjugated the refold-ed protein with group A meningococcal polysaccharide (GAMP) using the chemical method(ADH) ,the pol-ysaccharide-protein conjugates(GAMP-rCTB) were prepared. BALB/c mice were immunized either intraper-itoneally ( i. p. ) or intranasally ( i. n. ) with GAMP-rCTB. Moreover, GAMP-TT vaccine that TT as carrier proteins was i.n. immunized to the mice. The evaluation of immunology is performed. Results The conju-gates of polysaccharide-potein with the rCTB and TT as protein carrier both are able to elicit high level of GAMP specific IgG antibody in serum after i.n. immunization, and the conjugates can also elicit specific IgA antibody in lung lavage and intestinal mucosa. Conclusion rCTB and TT can both as the protein carri-er for polysaccharide-protein conjugate as mucosal vaccine. The route of intranasal may be more ways for im-mune function than i.p. immunization when rCTB is used as the carrier of the polysaccharide-protein conju-gates.