Search Results (418)

Background: Histone post-translational modifications (PTMs) play a pivotal role in the regulation of chromatin structure and gene expression, making them key targets in structural and epigenetic research. Synthetic histone peptides bearing specific PTMs are essential tools for elucidating the molecular mechanisms of histone function and protein–histone interactions. Methods: We synthesized histone H4 tail peptides containing site-specific lysine modifications using solid-phase peptide synthesis (SPPS). The correct synthesis of the peptides was confirmed by their molecular weights using a mass spectrometer. Results: An improved high-performance liquid chromatography (HPLC) method was developed to efficiently separate peptides with one modification difference. In alignment with green chemistry principles, we evaluated ethanol and isopropanol as an alternative organic solvent to acetonitrile in the mobile phase. The optimized HPLC method using acetonitrile enabled effective resolution of closely related peptide species, providing peptides suitable for downstream applications requiring high purities such as structural biology. Conclusions: This study presents a strategy for the purification of histone PTM peptides, emphasizing both analytical performance and sustainability. Further investigation must be undergone to develop high-precision purification using green chemicals. Full article

This study investigates the anti-obesity and antidiabetic potential of P. palmata extracts produced through sequential enzymatic and alkaline treatments. Among the treatment groups, the extract treated solely with Alcalase^® (Alc) demonstrated the highest protein content (10.11 ± 0.15%) and degree of hydrolysis (30.36 ± 0.77%), significantly outperforming other treatments (p < 0.05). The Alc extract also exhibited superior inhibitory activity against porcine pancreatic lipase and α-amylase, achieving the lowest IC₅₀ for lipase (2.29 ± 0.87 mg.mL⁻¹) and showing significant enzyme inhibition across all tested concentrations (p < 0.05). Ultrafiltration of the Alc extract revealed that peptide fractions < 1 kDa and 1–3 kDa were most effective in enzyme inhibition, with IC₅₀ values of 3.25–3.55 mg.mL⁻¹ for both lipase and α-amylase. Peptides were identified via LC-MS/MS analysis and database searching using SequestHT, resulting in 536 sequences, of which bioinformatic screening yielded 51 non-toxic, non-allergenic candidates (PeptideRanker score > 0.6); four of these contained known inhibitory motifs for lipase and α-amylase. Molecular docking confirmed strong binding affinities between these peptides and their respective enzymes, supporting their potential as natural enzyme inhibitors. These findings indicate the functional food potential of Alcalase^®-derived P. palmata peptides for managing obesity and type 2 diabetes. Full article

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEM_WT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His_6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His_6×-TCS-ΔNspSEM_WT. Unexpectedly, 4 °C desalting reduced the fusion protein’s molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R–G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H₁₇₂/S₁₇₈/D₂₁₂) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His_6×-TCS-ΔNspSEM_S178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity’s origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R–G bond cleavage. This work establishes a foundation for understanding ΔNspSEM_WT’s TLA. Full article

The prevalence of infectious diseases is steadily increasing. If left untreated, they can lead to more serious health problems. Antibiotics currently available on the market are facing growing resistance, prompting the development of increasingly powerful antibacterial molecules. One alternative currently under investigation is the use of antibacterial peptides, whose mechanisms of action differ from those of conventional drugs. These peptides are produced naturally by all living organisms and can also be synthesized. However, as peptide chains become longer, synthesis and purification become increasingly complex and laborious. For decades, antimicrobial peptides have been synthesized on polymer supports using automated systems. Unfortunately, longer chains tend to fold more, preventing access of reagents within the cross-linked polymer network. Recombinant production of antimicrobial peptides has been achieved in various organisms called “cell factories,” allowing for more sustainable synthesis. Recently, microalgae have emerged as a promising and sustainable alternative for the production of antimicrobial peptides. They are inexpensive, easy to cultivate, and capable of producing biologically valuable molecules, offering a potential solution to antibiotic resistance. This work reviews the current state of these “cell factories” and examines the advantages and limitations of microalgae for the future of biopharmaceutical production. Full article

Obesity is a worldwide problem, and lowering pancreatic lipase (PL) activity is an effective strategy to counteract it. In this study, pancreatic lipase-inhibitory (PL-I) peptides were isolated and purified from Chlorella pyrenoidosa protein hydrolysates (CPPHs) using ultrafiltration and Sephadex gel chromatography (Sephadex G-25). A total of 858 peptides were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Four novel PL-I peptides (FLSQPF, VWTPI, IVGPF, and IPYPL) were virtually screened using molecular docking and subsequently synthesized, with VWTPI exhibiting the highest PL inhibition. Moreover, the inhibition of the enzyme by VWTPI was a mixture of competitive and non-competitive inhibition, with an inhibition constant (Ki) of 7.27 mg/mL. Molecular docking showed that VWTPI interacts with the PL active center by hydrogen bonding, hydrophobic contacts, van der Waals forces, and π-π stacking. This study suggests that peptides from Chlorella pyrenoidosa could be used as lipid-lowering agents to prevent and cure obesity. Full article

Genetic fusion of a tag sequence to a target protein, or protein of interest (POI), is one of the most widely used technologies for recombinant expression. Tag-fusion proteins can enhance soluble expression, prolong half-life, increase binding avidity, and facilitate protein purification or refolding. In addition, tag-fusion proteins can be used to identify POI-binding partners through pull-down or immunoprecipitation assays. Beyond these classical applications, tags have evolved to serve as multifunctional tools, enabling real-time imaging, spatial localization, targeted delivery, and regulation of protein activity in living systems. Some engineered tags also allow conditional control, such as pH or ligand-dependent stabilization, thus expanding their utility in synthetic biology and therapeutic design. Here, we summarize protein-based and peptide-based tags, as well as methods for tag removal. While not fully comprehensive, this review aims to help researchers design suitable tag formats for specific goals. Full article

Background and Methods: To address the need for dietary interventions in sub-healthy populations and promote sustainable utilization of agricultural by-products, we isolated Pinus pumila hypoglycemic peptide (PHP) from nut oil meal through enzymatic extraction, ion exchange and gel chromatography purification, and simulated gastric digestion. Results: PHP exhibited significant inhibitory activity against α-amylase and α-glucosidase. In type 2 diabetic mice, PHP significantly ameliorated the “three-more-one-less” syndrome, reduced glycosylated hemoglobin and insulin levels, mitigated liver and kidney tissue lesions, and improved glucose and lipid metabolic disorders—effects partly supported by its enhancement of intestinal barrier function via restoring gut microbiota diversity. Gut microbiota analysis revealed that PHP exerts hypoglycemic effects by regulating gut microbial composition: increasing SCFA-producing taxa, reducing pro-inflammatory/metabolic disorder-associated taxa, and normalizing the Firmicutes/Bacteroidetes ratio. KEGG pathway analysis demonstrated that PHP mediates synergistic hypoglycemic effects by regulating carbohydrate metabolism, amino acid metabolism, and cofactor/vitamin metabolism. Conclusions: This work provides a theoretical foundation for developing natural functional foods from agricultural by-products, supporting PHP’s potential as a dietary supplement for metabolic regulation. Full article

Background/Objectives: Listeria monocytogenes is a high-risk pathogen in the food industry involved in several outbreaks. Bacteriocins are natural-origin antimicrobial peptides or proteins that represent a good alternative to synthetic antimicrobials capable of inhibiting the growth of pathogens. This study aimed to purify and identify bacteriocins from the cell-free supernatant of Enterococcus lactis-67, which exhibits antagonistic activity against L. monocytogenes. Methods: Protein purification was performed by precipitation with ammonium sulfate, dialysis, and fast protein liquid chromatography. Active protein fractions were analyzed by SDS-PAGE and identified by mass spectrometry. Results: In addition to enterocins A and B, a novel 47 kDa bacteriocin with LysM and NlpC/P60 domains, on the N- and C-terminal regions, respectively, was identified. This enterocin has not been described for Enterococcus before. Conclusions: This study contributes to the identification of new natural and effective strategies for ensuring food safety. Full article

Polypeptides exhibit significant health-promoting effects through diverse biological activities, including antihypertensive, antidiabetic, anti-cancer, antimicrobial, and antioxidant properties. Membrane technology offers an efficient separation approach for polypeptides due to its high efficiency, low energy consumption, operational simplicity, and environmental sustainability. This review briefly described the advancements in membrane separation of polypeptides and highlighted the major implementation challenges, such as membrane fouling, peptide adsorption losses, and compromised separation efficiency caused by peptide aggregation. Contributing factors for each issue based on the progress and reports of relevant research were analyzed. And solutions and strategies were also summarized as feed pretreatment, operational parameter optimization, aggregate elimination, and membrane surface modification. These approaches could reduce product loss and enhance peptide yield during purification. This review can provide reference for the research on efficient membrane separation of polypeptide products. Full article

The advancement of photocatalytic materials is critical for addressing environmental challenges such as water remediation, where efficient, robust, and reusable systems are in high demand. In this search, the development of hierarchically organized photocatalytic configurations with spatial control over active sites can significantly enhance performance. With this in mind, we present here a novel biomimetic approach for the patterned growth of TiO₂-ZnO photocatalytic heterostructures using solid-binding peptides (SBPs) as molecular linkers. Specifically, using bi-functional SBPs with selective affinity for both oxides, we achieve site-specific, molecularly guided deposition of TiO₂ nanoparticles onto pre-patterned ZnO-coated substrates. Leveraging the specific recognition capabilities and strong binding affinities of the engineered SBPs, the proposed biomimetic methodology allows for the fabrication of well-organized hybrid nanostructures under sustainable conditions. Photocatalytic degradation assays employing methyl orange as a model contaminant indicate that the patterned architecture enhances both the accessibility of the active photocatalytic sites and the recoverability of the material. This reusability is a critical parameter for the practical deployment of photocatalytic systems in water purification technologies. The obtained results underscore the potential of SBP-mediated molecular recognition as a versatile tool for green nanofabrication of functional materials with advanced architectural and catalytic properties. Full article

This study presents a strategy to develop crayfish shell peptides with enhanced antioxidant and angiotensin-I-converting enzyme (ACE) inhibitory properties. Crayfish shell protein hydrolysates (CSPH1–3) with different molecular weights were analyzed. CSPH2 (3–5 kDa) exhibited the strongest antioxidant activities, which could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the 2,2′-azobis(3-ethylbenzothiazoline-6-sulfonic acid) sodium salt (ABTS) radical by (77.40 ± 4.54)% and (91.59 ± 0.30)%, respectively, and ACE inhibition activity of (64.74 ± 0.64)%. CSPH2 was further separated into three fractions, and CSPHF2 showed the maximum biological activity. The sequences of the purified antioxidant peptide (APAPLPPPAP) and ACE inhibitory peptide (QGPDDPLIPIM) were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in CSPHF2. These peptides increased the nitric oxide (NO) concentration and decreased the endothelin-1 (ET-1) content in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, while also inhibiting reactive oxygen species (ROS). In addition, CSPH showed protective effects in terms of oxidative damage to HepG2 cells induced by H₂O₂. These findings suggest that crayfish shell peptides have potential applications as ingredients in antihypertensive agents and antioxidants, offering significant health benefits when consumed. Full article

Sesame oil extraction byproduct (SOEB) contains a high percentage of protein (49.81 g/100 g), making it a suitable plant-based source for producing protein hydrolysates with nutraceutical potential. In this study, albumins, globulins, glutelins, and prolamins fractions were extracted and characterized from SOEB. These fractions were then enzymatically hydrolyzed with alcalase, yielding high soluble protein content (>90%) and hydrolysis degrees ranging from 34.66 to 45.10%. The hydrolysates were fractionated by molecular weight (<5 kDa, 3–5 kDa, 1–3 kDa, and <1 kDa). These fractions demonstrated potential for inhibiting the DPPH radical (25.19–95.79%) and the α-glucosidase enzyme (40.14–55.63%), particularly the fractions with molecular weight <1 kDa. We identified 28 peptides, with molecular weights between 332.20 and 1096.63 Da, which showed potent antioxidant activities (IC₅₀ = 90.18 µg/mL), as well as inhibitory effects on key enzymes such as α-glucosidase (IC₅₀ = 61.48 µg/mL), dipeptidyl peptidase IV (IC₅₀ = 12.12 µg/mL), and pancreatic lipase (IC₅₀ = 6.14 mg/mL). These results demonstrate the antioxidant, antihyperglycemic, antidiabetic, and anti-obesity potential of SOEB peptides, highlighting their use in the formulation of new functional foods or nutraceuticals. Full article

Twelve undescribed peptidic compounds, bukhansantaibols A–K (1–10) and bukhansantaibals A–B (11–12), were isolated from the soil fungus Trichoderma atroviride through LC-MS and bioactivity-guided purification. Their structures were elucidated by the analysis of 1D and 2D NMR spectra, HRESIMS, and acid hydrolysis using modified Marfey’s method. All compounds were evaluated for their cytotoxic activity against HCT-8 (colon cancer) and SK-OV-3 (ovarian cancer) cells. Among them, compounds 1–5 exhibited significant inhibitory effects, with IC₅₀ values ranging from 2.1 to 19.6 μM. Full article

Proximity biotinylation, which utilizes various biotin ligating enzymes (BioID, TurboID, etc.), is widely used as a powerful tool for identifying novel protein–protein interactions. However, this method has a significant limitation: the use of streptavidin on beads for enriching biotinylated proteins often results in a high background of peptides from streptavidin itself, which interferes with identification by peptide mass fingerprinting. This limitation makes it practically impossible to study samples containing a small amount of material, such as individual insect tissues. In this study, we compared different precipitation and elution conditions for the purification of biotinylated proteins from protein extracts of Drosophila melanogaster S2 cells. We found that biotinylated proteins can be purified using anti-biotin antibodies, although with lower efficiency than streptavidin-based resin. We also demonstrated that protease-resistant streptavidin (prS), previously tested in mammalian cells, can be used effectively to purify biotinylated proteins from tissues of D. melanogaster. In our experiments, prS showed precipitation efficiency comparable to regular streptavidin but generated a lower background in peptide fingerprinting. To further demonstrate the applicability of prS for studying protein–protein interactions in D. melanogaster tissues, we carried out experiments to identify interaction partners of the ecdysone receptor (EcR) in D. melanogaster ovarian tissue using TurboID-based proximity biotinylation. As a result, EcR was found to interact with both previously described and novel protein partners in Drosophila ovaries. Full article

Background: With the increasing shift in drug design away from classical drug targets towards the modulation of protein-protein interactions, synthetic peptides are gaining increasing relevance. The synthesis and purification of peptides via solid-phase peptide synthesis (SPPS) strongly rely on trifluoroacetic acid (TFA) as a cleavage agent and ion-pairing reagent, respectively, resulting in peptides being obtained as TFA salts. Although TFA has excellent properties for peptide production, numerous studies highlight the negative impact of using peptides from TFA⁻ salts in biological assays. Methods: Investigated peptides were synthesized via SPPS and the TFA⁻ counterion was exchanged for Cl⁻ via freeze-drying in different concentrations of HCl. Detection and quantification of residual TFA⁻ were carried out via FT-IR, ¹⁹F-NMR, and HPLC using an evaporative light-scattering detector (ELSD). A liposomal fluorescence assay was used to test for the influence of the counterion on the peptides’ passive membrane permeability. Results: All TFA⁻ detection methods were successfully validated according to ICH guidelines. TFA⁻ removal with 10 mM HCl was determined to be the optimal condition. No impact on peptide purity was observed at all HCl concentrations. Influences on permeability coefficients depending on peptide sequence and salt form were found. Conclusions: This study presents a systematic investigation of the removal of TFA⁻ counterions from synthetic peptides and their replacement with Cl⁻ counterions. Detected counterion contents were used to understand the impact of sequence differences, especially positive charges, on the amount and potential localization of counterions. Our findings emphasize the importance of counterion quantification and specification in assays with synthetic peptides. Full article