The protein journal最新文献

In this work, studies were conducted to achieve the complete extraction of sericin from natural silk cocoons at temperatures of 100 °C and 110 °C for durations ranging 2-24 h without the use of chemical reagents. As a result, sericin samples with molecular weight changing 20-240 kDa were obtained. During the thermal and sublimation drying of the isolated sericin samples, changes in their solubility were observed. Structural changes in the thermally dried sericin samples were analyzed using X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. XRD analysis revealed diffraction peaks at 18.9° and 20.7°, indicating the presence of crystalline structure, whereas the samples dried by sublimation exhibited a completely amorphous structure. FTIR spectroscopy showed band broadening in the regions of 3000-3500 cm^- 1 and 1500-1600 cm^- 1 in the thermally dried samples, which was attributed to extensive hydrogen-bond formation compared to the samples dried by sublimation. Rheological analysis further demonstrated that the loss modulus exceeded the storage modulus for sericin sample. The results demonstrate that extract sericin under high temperature without chemical reagent enables the efficient isolation of sericin with different molecular weight, while the drying method strongly influences its structural, solubility, and rheological properties.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major cause of difficult-to-treat infections, largely due to its multidrug resistance and capacity to form persistent biofilms on medical and industrial surfaces. Bacteriophage-derived endolysins have emerged as promising antibacterial agents, but many still require detailed biochemical characterization to support their potential applications. The present study describes the staphylococcal endolysin MV-L expression, purification, and functional analyses, evaluating its ability to control MRSA on surfaces and in biofilms. MV-L was recombinantly produced in Escherichia coli, purified by nickel affinity chromatography; its lytic activity against exponentially growing MRSA was assessed under different physicochemical conditions. Enzyme exhibited optimal activity in slightly alkaline conditions and moderate temperatures; its performance was strongly influenced by ionic strength and divalent cations. Under optimized conditions, MV-L showed markedly increased lytic efficiency and retained activity at low protein concentrations. Beyond planktonic cells, MV-L significantly reduced MRSA loads on glass and stainless steel and disrupted pre-formed biofilms on polystyrene. These findings highlight how buffer composition and ion availability critically modulate MV-L activity and support the concept that tailored endolysins can be integrated as complementary strategies for MRSA control, particularly in scenarios where conventional disinfectants and antibiotics are limited by resistance or poor efficacy against biofilms.

Arginine is widely used in protein formulations to suppress protein aggregation and prolong the lifetime of the native, functional state which undergoes different stresses during storage, shipping and handling. However, arginine's differential effects on protein aggregation pathways remain unclear. Here, we investigated the effects of arginine on heat- and acid-induced aggregation of immunoglobulin G and heat-induced aggregation of several model proteins. Arginine suppressed the formation of insoluble aggregates but had limited impact on monomer recovery, particularly on immunoglobulin G. Instead, arginine promoted the formation of soluble aggregates, suggesting a possibility that unfolded proteins possess heterogeneous surface properties, leading to formation of soluble and insoluble aggregates that are modulated by arginine. To explain these kinetic behaviors, we propose a hierarchical aggregation model, in which arginine preferentially modulates weaker intermolecular interactions to inhibit the growth of the soluble aggregates into large insoluble aggregates. Moreover, soluble aggregates were observed in a range of proteins with varying isoelectric points and molecular weights. These results highlight a previously unrecognized aspect of soluble protein aggregation. Understanding the pathway and mechanism of soluble aggregate formation may shed light into physiologically-relevant soluble assemblies, e.g., Alzheimer's disease-associated oligomers and microtubule-derived double rings in contrast to the soluble amorphous aggregates under current study.

The presence of tumor heterogeneity is a critical issue that restricts the success of targeted therapies and negatively impacts patient outcomes. Recent studies have concentrated on the development of multi-epitope peptides that exhibit considerable overexpression in cancerous tissues with the aim of activating immune cells and utilizing immune-mediated responses to effectively suppress tumor growth. In this study, genes with increased expression in colorectal cancer (CRC) were identified using GEO data and R software. Following overexpression confirmation of APCDD1, we identified epitopes from the protein that can be recognized by various MHC molecules and presented on APC cell surfaces. Subsequent to expression and purification of the multi-epitope peptide and investigation on the BALB/c mice harboring tumor xenograft, obtained results showed a significant reduction in tumor growth, mitotic cell count, angiogenesis, metastasis, and an increase in Tumor-Infiltrating Lymphocytes (TILs) in the tumor microenvironment. Overall, the finding highlight the potential of multi-epitope peptide in CRC immunotherapy, where it may address the significant challenge of tumor heterogeneity.

Bacterial alpha-amylases have diverse industrial applications in food, fermentation, and pharmaceuticals. This study focuses on the isolation and characterization of a novel alpha-amylase-producing bacterium through molecular and in silico analyses, including molecular docking to determine enzyme-substrate specificity and binding interactions. Among nine bacterial isolates, S4 demonstrated the highest amylolytic activity of 63.68 U/ml. Molecular identification revealed isolate (S4) identity as Bacillus subtilis (OM278386). Enzyme charcterization revealed that maximum enzyme activity was observed at 40 °C and pH 7.0, after 24 h. The full-length novel alpha-amylase gene from B. subtilis (S4) was amplified, sequenced, and translated into a protein sequence. A putative protein was subjected to BLASTp, phylogenetic analysis, and physicochemical characterization. A 3D model was generated and validated through homology modeling. Molecular docking was performed using six substrates: amylopectin, maltotetraose, glycogen, starch, amylose, and cyclodextrin to determine substrate specificity. The putative AmyE protein comprised 488 amino acids. Phylogenetic analysis confirmed its close association with alpha-amylases of other Bacillus species. The enzymes exhibited industrially desirable traits, including high stability, thermotolerance, and hydrophilicity. In contrast, 3D model investigation showed excellent stereochemical quality, with 95.2% of amino acids in the favored region of the Ramachandran plot. Docking studies revealed the highest affinity for amylopectin (binding energy: - 7.2 kcal/mol). Two essential amino acid residues, Asp and Glu-318, were identified as crucial for active-site substrate interactions and enzyme catalysis across various substrates. In conclusion, the analysis presents alpha-amylase from B. subtilis stain S4 as a promising candidate for diverse industrial applications, offering cost-effective alternatives for starch processing, food preservation, and other biotechnological processes.

SARS-CoV-2 consists of the spike (S) protein which plays an important role in mediating the entry of virus into the host and it mainly consists of two subunits which are functionally different from each other. The first subunit S1, is involved in binding to the host receptor such as ACE2, and the second subunit S2, is involved in facilitating the viral membrane fusion with that of host membrane. Despite extensive research on the S1 domain due to its immunodominance and variability, the structurally conserved S2 domain remains relatively understudied. Recognizing the potential of S2 in modulating host responses, this study focuses on its recombinant expression, purification, and functional impact. The S2 coding region was cloned into the pGEX2TK plasmid to produce protein, which is a GST fusion-based protein and thereafter, the expression was done in BL21(DE3) strain of Escherichia coli cells. To enhance yield as well as solubility, protein expression was induced at a reduced temperature of 16 °C, minimizing aggregation and degradation. The fusion protein was purified via glutathione affinity chromatography, yielding high-purity S2 suitable for downstream applications. The S2 protein upon transfection into HEK293 and WI-38 mammalian cells leads to the expression of downregulated insulin-like growth factor 1 receptor (IGF-1R), as measured with the help of protein analysis. In order to highlight the role in pathogenesis of COVID-19 through modulation of cellular receptor and for the intervention of therapeutics, S2 can likely be considered a potential target.

D-p-hydroxyphenyl glycine (D-PHPG) is a D-amino acid used as an intermediate in the synthesis of semi-synthetic antibiotics. It is synthesized from hydantoin derivatives through two sequential enzymatic reactions involving D-hydantoinase (D-hase) and D-carbamoylase (D-case). Although whole-cell biocatalysis of D-PHPG is cost-effective, its efficiency suffers from transport obstacles, intracellular degradation, and limited substrate solubility. This study utilized a bacterial surface display system to express D-hase and D-case in Escherichia coli for D-PHPG production. Enzyme production optimization was carried out in two stages. Initially, key factors influencing cell density during co-culture were identified through culture media and fermentation parameters screening using the Plackett-Burman design, followed by optimization with the D-optimal method. Next, induction parameters were fine-tuned using response surface methodology. The optimal culture medium was found to contain glycerol (12 g/L) and yeast extract (15 g/L) under optimal induction conditions (0.17 mM IPTG, OD₆₀₀ of 1.3, and 21 °C). These conditions achieved an OD₆₀₀ of 15.6, with expression levels of 20.18% for D-case and 20.82% for D-hase. Scaling up in a stirred tank bioreactor resulted in an OD₆₀₀ of 32.15, with D-hase and D-case expression levels increasing to 25.8 and 24.2%, respectively, and enzymatic activities improving by 2.83 times for D-case and 3.42 times for D-hase. The optimized co-culture approach under optimized induction conditions achieved a conversion yield of 95% and a D-PHPG production yield of 90%. The study results showed that the suggested fermentation conditions will contribute to future scale-up studies aimed at improving enzyme activities for other surface protein production.