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ACS Omega Oct 2021Platelet adhesion and denaturation on artificial medical implants induce thrombus formation. In this study, bioabsorbable copolymers composed of...
Platelet adhesion and denaturation on artificial medical implants induce thrombus formation. In this study, bioabsorbable copolymers composed of poly(l-lactide--glycolide) (PLGA) and poly(1,5-dioxepan-2-one) (PDXO) were synthesized and evaluated for their antiplatelet adhesive properties. The PLGA-PXO multiblock copolymer (PLGA-PDXO MBC) and its random copolymer (PLGA-PDXO RC) showed effective antiplatelet adhesive properties, and the number of adhered platelets was similar to those adhered on poly(2-methoxyethylacrylate), a known antiplatelet adhesive polymer, although a large number of denatured platelets were observed on a PLGA-poly(ε-caprolactone) multiblock copolymer (PLGA-PCL MBC). Using monoclonal antifibrinogen IgG antibodies, we also found that both αC and γ-chains, the binding sites of fibrinogen for platelets, were less exposed on the PLGA-PDXO MBC surface compared to PLGA-PCL MBC. Furthermore, free-standing films of PLGA-PDXO MBC were prepared by casting the polymer solution on glass plates and showed good tensile properties and slow hydrolytic degradation in phosphate-buffered saline (pH = 7.4). We expect that the unique properties of PLGA-PDXO MBC, i.e., antiplatelet adhesive behavior, good tensile strength, and hydrolytic degradation, will pave the way for the development of new bioabsorbable implanting materials suitable for application at blood-contacting sites.
PubMed: 34722996
DOI: 10.1021/acsomega.1c03846 -
Advanced Biomedical Research 2020is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections....
BACKGROUND
is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate.
MATERIALS AND METHODS
In this study, the fusion protein () and full genes () were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 coli. Overlap polymerase chain reaction (PCR) using different primers for 5' and 3' end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning.
RESULTS
Cloning of both and into the pET28a vector and their transform into the BL21 (DE3) host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration.
CONCLUSION
Changing the vector to pBAD/gIII A and consequently changing the host to Top10 have resulted in sufficient expression, which shows that Top10 may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.
PubMed: 33072655
DOI: 10.4103/abr.abr_245_19 -
Molecules (Basel, Switzerland) Feb 2022Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are... (Review)
Review
Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.
Topics: Animals; Cattle; Lactoglobulins; Milk; Models, Molecular; Protein Denaturation; Protein Stability; Protein Unfolding; Thermodynamics
PubMed: 35164393
DOI: 10.3390/molecules27031131 -
Analytical Chemistry Feb 2023The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for...
The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.
Topics: Humans; Protein Denaturation; Serum Albumin, Bovine; Hot Temperature; Fluorescence Polarization
PubMed: 36696963
DOI: 10.1021/acs.analchem.2c03912 -
Scientific Reports Mar 2023Marine elasmobranch fish contain urea, a protein denaturant, in their bodies. The urea-trimethylamine N-oxide (TMAO) counteraction mechanism contributes to...
Marine elasmobranch fish contain urea, a protein denaturant, in their bodies. The urea-trimethylamine N-oxide (TMAO) counteraction mechanism contributes to urea-resistibility, where TMAO compensates for protein denaturation by urea. However, previous studies revealed that shark fast skeletal muscle myosin exhibits native activity at physiological urea concentrations in the absence of TMAO, suggesting that shark myosin has urea-resistibility. In this study, we compared the urea-resistibility of myosin alkali light chains (A1-LC and A2-LC) from banded houndshark and carp by examining the α-helical content at various urea concentrations. The α-helical content of carp myosin A1-LC and A2-LC gradually decreased as urea concentrations increased to 2 M. In contrast, the α-helical content of banded houndshark A1-LC increased between 0 and 0.5 M urea, and the α-helical content of A2-LC remained constant until 0.5 M urea. We determined the full-length sequences of the banded houndshark myosin light chains (A1-LC, A2-LC and DTNB-LC). Hydrophilicity analysis revealed that the N-terminal region (residues 28-34) of A1-LC from banded houndshark is more hydrophilic than the corresponding region of A1-LC from carp. These findings support the notion that shark myosin exhibits urea-resistibility independent of the urea-TMAO counteraction mechanism.
Topics: Animals; Myosin Light Chains; Urea; Sharks; Muscle, Skeletal
PubMed: 36966252
DOI: 10.1038/s41598-023-32228-w -
ACS Omega Nov 2022The main challenge for the preparation of protein single-chain nanoparticles (SCNPs) is the natural complexity of these macromolecules. Herein, we report the suitable...
The main challenge for the preparation of protein single-chain nanoparticles (SCNPs) is the natural complexity of these macromolecules. Herein, we report the suitable conditions to produce "neat" bovine serum albumin (BSA) single-chain nanoparticles (SCNPs) from partially denatured BSA, which involves denaturation in urea and intramolecular cross-linking below the overlap concentration. We use two disuccinimide ester linkers containing three and six methylene spacer groups: disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS), respectively. Remarkably, the degree of internal cross-linking can be followed simply and efficiently via H NMR spectroscopy. The associated structural changes-as probed by small-angle neutron scattering (SANS)-reveal that the denatured protein has a random-like coil conformation, which progressively shrinks with the addition of DSG or DSS, thus allowing for size control of the BSA-SCNPs with radii of gyration down to 5.4 nm. The longer cross-linker exhibits slightly more efficiency in chain compaction with a somewhat stronger size reduction but similar reactivity at a given cross-linker concentration. This reliable method is applicable to a wide range of compact proteins since most proteins have appropriate reactive amino acids and denature in urea. Critically, this work paves the way to the synthesis of "neat", biodegradable protein SCNPs for a range of applications including nanomedicine.
PubMed: 36440132
DOI: 10.1021/acsomega.2c04805 -
Journal of Food Science and Technology Feb 2023In this study, the denatured whey protein paste (DWPP) was used to improve the texture characterizations of Gouda cheese. Five treatments of cheese were manufactured by...
In this study, the denatured whey protein paste (DWPP) was used to improve the texture characterizations of Gouda cheese. Five treatments of cheese were manufactured by adding 0, 1, 2, 3 and 4% of DWPP to cheese curd. Fortification of Gouda cheese with DWPP increased values of moisture, salt in moisture, water-soluble nitrogen/total nitrogen and non-protein nitrogen/total nitrogen whereas decreased values of density and free oil. The cheese contained DWPP was lighter and more yellowish compared to the control. The cheese samples contained 1 and 2% DWPP exhibited a significant increase in hardness, cohesiveness, springiness, gumminess and chewiness values while, the cheese samples that contained 3 and 4% DWPP exhibited a significant decrease. Adding DWPP to cheese lowered saturated fatty acids and raised unsaturated fatty acid (USFA) values which partially caused a lowering in cheese hardness at high levels of DWPP because of the low melting points of USFA. Based on these results, supplementation of Gouda cheese with 1 or 2% DWPP improved the texture properties.
PubMed: 36712195
DOI: 10.1007/s13197-022-05643-6 -
Journal of Medicinal Chemistry Apr 2021Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two...
Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two mechanisms might explain this: low concentrations of the colloid and enzyme might mean low encounter rates, or colloid-based protein denaturation might impose a kinetic barrier. These two mechanisms should have different concentration dependencies. Perplexingly, when enzyme concentration was increased, incubation times actually lengthened, inconsistent with both models and with classical chemical kinetics of solution species. We therefore considered molecular crowding, where colloids with lower protein surface density demand a shorter incubation time than more crowded colloids. To test this, we grew and shrank colloid surface area. As the surface area shrank, the incubation time lengthened, while as it increased, the converse was true. These observations support a crowding effect on protein binding to colloidal aggregates. Implications for drug delivery and for detecting aggregation-based inhibition will be discussed.
Topics: Adsorption; Bacterial Proteins; Colloids; Enzyme Assays; Fulvestrant; Kinetics; Malate Dehydrogenase; Protein Binding; Sorafenib; beta-Lactamases
PubMed: 33761256
DOI: 10.1021/acs.jmedchem.0c02253 -
Proceedings of the National Academy of... Apr 2024Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known...
Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known denaturant, and TMAO is a protective osmolyte that counteracts urea-induced protein denaturation. TMAO also has a general protein-protective effect, for example, it counters pressure-induced protein denaturation in deep-sea fish. These opposing effects on protein stability have been linked to the spatial relationship of TMAO, urea, and protein molecules. It is generally accepted that urea-induced denaturation proceeds through the accumulation of urea at the protein surface and their subsequent interaction. In contrast, it has been suggested that TMAO's protein-stabilizing effects stem from its exclusion from the protein surface, and its ability to deplete urea from protein surfaces; however, these spatial relationships are uncertain. We used neutron diffraction, coupled with structural refinement modeling, to study the spatial associations of TMAO and urea with the tripeptide derivative glycine-proline-glycinamide in aqueous urea, aqueous TMAO, and aqueous urea-TMAO (in the mole ratio 1:2 TMAO:urea). We found that TMAO depleted urea from the peptide's surface and that while TMAO was not excluded from the tripeptide's surface, strong atomic interactions between the peptide and TMAO were limited to hydrogen bond donating peptide groups. We found that the repartition of urea, by TMAO, was associated with preferential TMAO-urea bonding and enhanced urea-water hydrogen bonding, thereby anchoring urea in the bulk solution and depleting urea from the peptide surface.
Topics: Animals; Urea; Peptides; Water; Methylamines; Membrane Proteins
PubMed: 38536756
DOI: 10.1073/pnas.2317825121 -
International Journal of Molecular... Aug 2022Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform...
Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications.
Topics: Animals; Caco-2 Cells; Disulfides; Gastrointestinal Tract; Horseradish Peroxidase; Humans; Mice; Nanoparticles
PubMed: 36077259
DOI: 10.3390/ijms23179856