Protein purification is a critical prerequisite for biochemical, biophysical, and structural analyses. Although proteins can be isolated from their native environments, this approach is often limited by low yield and heterogeneity. Recombinant protein expression provides an efficient alternative, allowing the production of large quantities of target proteins in diverse host systems. To overcome challenges in yield, solubility, stability, and recovery, fusion tags are widely employed. These tags not only improve expression levels but also facilitate purification through affinity-based methods, and in some cases, contribute directly to functional or structural characterization. This review provides an overview of commonly used fusion tags, and considerations for selecting appropriate tags based on experimental needs and the target proteins. By enabling streamlined expression and purification, fusion tags expand opportunities to investigate protein structures, folding, and interaction networks. Such advances ultimately enhance our understanding of protein function and support the rational design of novel therapeutics.
Argininosuccinate synthase 1 (ASS1) is a key rate-limiting enzyme in the urea cycle and is highly conserved at the protein level. Its catalytic activity directly determines the efficiency of ammonia clearance. Hyperammonemia is a clinical syndrome caused by abnormally elevated blood ammonia levels and is characterized mainly by central nervous system dysfunction, with neurotoxicity and a high mortality rate. When liver function is impaired or key enzymes of the urea cycle are deficient, ammonia clearance is reduced, leading to ammonia accumulation and passage across the blood-brain barrier, resulting in disturbances of consciousness, coma, or even death. Among currently used ammonia-lowering agents, L-ornithine-L-aspartate (LOLA) can promote the urea cycle to reduce blood ammonia levels and protect the liver; however, high doses are prone to gastrointestinal adverse effects, limiting its clinical application. This study obtained biologically active human ASS1 protein through prokaryotic expression and analyzed its enzymatic kinetic properties, aiming to explore the ammonia-lowering effect of ASS1 combined with LOLA and to provide new ideas and potential enzymatic intervention strategies for the clinical treatment of hyperammonemia. The full-length open reading frame of human ASS1 was amplified by reverse transcription polymerase chain reaction (RT-PCR), and both the PCR product and the pET-28a vector were digested with restriction enzymes. After purification by agarose gel electrophoresis, the pET-28a-ASS1 prokaryotic expression vector was successfully constructed using homologous recombination technology. The recombinant plasmid was transformed into Escherichia coli, and expression of the 6×His-ASS1 recombinant protein was induced by isopropyl β-D-thiogalactopyranoside (IPTG). The protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. In vitro enzyme activity assays were performed to verify the biological activity of the recombinant protein. An enzymatic reaction system using citrulline (Cit) and aspartate (Asp) as substrates was established to determine the optimal temperature and pH conditions for ASS1 activity and to further analyze its enzymatic kinetic parameters. A hyperammonemia mouse model was established in C57BL/6 mice by intraperitoneal injection of NH4Cl (400 mg/kg). The ammonia-lowering effects of ASS1 combined with LOLA were evaluated. Mice were randomly grouped, and 4 hours after modeling, ASS1, LOLA, or combined treatment was administered via the tail vein for 4 hours, followed by measurement of blood ammonia and urea levels. In addition, normal C57BL/6 mice were treated with combined ASS1 and LOLA for 24 hours, after which liver and renal function indices were assessed. The pET28a-ASS1 vector was successfully constructed using homologous recombination, and soluble recombinant ASS1 protein was obtained in a prokaryotic expression system. After purification and concentration by Ni-NTA affinity chromatography, the protein purity reached 95.4%±1.2%, with a concentration of (10.5± 0.8) mg/mL. In vitro enzyme activity assays systematically characterized the enzymatic properties of ASS1, showing an optimal reaction temperature of 37 ℃ and an optimal pH of 8.0. Using Asp and Cit as substrates, the enzymatic kinetic parameters were determined. The Km and kcat/Km values of ASS1 for Asp and Cit were (31.00±1.24) μmol/L and 2.07 L/(μmol·s), and (46.53±2.75) μmol/L and 1.35 L/(μmol·s), respectively. In vivo experiments showed that in an acute hyperammonemia mouse model induced by intraperitoneal injection of NH4Cl (400 mg/kg), ASS1 (50 mg/kg) reduced blood ammonia levels by 48.86% by accelerating the urea cycle. When ASS1 (50 mg/kg) was combined with low-dose LOLA (50 mg/kg), the ammonia-lowering efficiency was further enhanced, with a reduction of 76.31%, restoring blood ammonia levels to the normal range in hyperammonemic mice. Recombinant ASS1 protein obtained by prokaryotic expression exhibits stable and well-defined biological activity, with catalytic function significantly influenced by temperature and pH and displaying clear enzymatic kinetic characteristics. In an NH4Cl-induced mouse model of hyperammonemia, combined application of ASS1 and LOLA showed a more stable and effective ammonia-lowering effect than monotherapy, demonstrating good synergistic effects. These findings provide a new enzymatic intervention strategy for the treatment of hyperammonemia, lay a theoretical foundation for the development of combination ammonia-lowering therapies, and indicate potential application prospects. 目的: 精氨酸代琥珀酸合成酶1(argininosuccinate synthase 1，ASS1)是尿素循环的关键限速酶且蛋白质高度保守，其催化活性直接决定血氨的清除效率。高氨血症(hyperammonemia)是血氨异常升高引起的、以中枢神经系统功能障碍为主要表现的临床综合征，具有神经毒性和较高病死率。肝功能受损或尿素循环关键酶缺陷时，清除血氨能力下降，导致血氨蓄积并透过血脑屏障，引发意识障碍、昏迷甚至死亡。目前临床常用降氨药物中，门冬氨酸鸟氨酸(L-ornithine-L-aspartate，LOLA)可促进尿素循环降低血氨并保护肝，但高剂量易致胃肠道不良反应，限制其应用。本研究通过原核表达获得具有生物活性的人源ASS1蛋白，解析其酶促动力学特性，旨在探讨ASS1联合LOLA的降血氨作用，为临床高氨血症的治疗提供新的思路和潜在的酶学干预策略。方法: 通过逆转录聚合酶链式反应(reverse transcription polymerase chain reaction，RT-PCR)扩增获得人源ASS1的开放阅读框全长，并对该PCR产物及pET-28a载体进行酶切。经琼脂糖凝胶电泳回收纯化上述片段后，利用同源重组技术成功构建了pET-28a-ASS1原核表达载体。随后转化至大肠杆菌中表达并使用异丙基-β-D-硫代半乳糖苷(isopropyl β-D-thiogalactopyranoside，IPTG)诱导6×His-ASS1重组蛋白表达，采用镍柱亲和层析(nickel-nitrilotriacetic acid，Ni-NTA)进行蛋白质纯化。通过体外酶活实验验证重组蛋白的生物活性，构建以瓜氨酸(citrulline，Cit)和天冬氨酸(aspartate，Asp)为底物的酶促反应体系，测定ASS1的最适温度和最适pH反应条件，并进一步解析其酶促动力学参数。构建NH₄Cl(腹腔注射400 mg/kg)诱导的C57BL/6小鼠高氨血症模型，检测ASS1联合LOLA降血氨作用，随机分组，建模4 h后经尾静脉给予ASS1、LOLA或联合干预4 h，并检测血氨与尿素水平;另设ASS1和LOLA联合用药对C57BL/6正常小鼠给药24 h后检测肝功能及肾功能指标。结果: 采用同源重组技术成功构建并获得pET28a-ASS1载体，并在原核表达系统中获得可溶性重组蛋白ASS1;经Ni-NTA亲和层析纯化和浓缩后，蛋白质纯度达95.4%±1.2%，浓度为(10.5±0.8) mg/mL。在体外酶活实验中系统解析了ASS1的酶促反应特性，显示ASS1酶促反应的最适温度为37 ℃，最适pH值为8.0;结合底物Asp和Cit测定其酶促动力学参数，ASS1与Asp和Cit的Km和kcat/Km分别为(31.00±1.24) μmol/L，2.07 L/(μmol·s)和(46.53±2.75) μmol/L，1.35 L/(μmol·s);在体内实验中，通过腹腔注射400 mg/kg NH4Cl建立急性高氨血小鼠模型，ASS1(50 mg/kg)可通过加速尿素循环降低血氨浓度，使血氨浓度下降48.86%;ASS1(50 mg/kg)与低剂量LOLA(50 mg/kg)联合给药时，降氨效率进一步提升，血氨浓度下降76.31%，并可使高氨血症小鼠的血氨水平恢复至正常范围。结论: 通过原核表达获得的重组蛋白ASS1具有稳定且明确的生物活性，其催化功能受温度和pH条件显著影响，并呈现出清晰的酶促反应动力学特征。在NH₄Cl诱导的小鼠高氨血症模型中，ASS1与LOLA联合应用较单独用药表现出更稳定的降血氨效果，显示出良好的协同作用，为高氨血症的治疗提供了新的酶学干预思路，进而为发展联合降血氨策略奠定了理论依据，并具有潜在应用前景。.
There are various chitin-binding proteins in insects. Among them, the CPAP3 protein family containing the chitin-binding domain ChtBD2 is one of the most abundant types in the epidermis. This study aims to use insect chitin-binding proteins as tag proteins to explore their application potential in the field of protein purification, and simultaneously investigate the relationship between the number of domains of CPAP3 protein and its binding capacity. CPAP3-A (abbreviated as CPAP3) from silkworm was used as the tag protein, and enhanced green fluorescent protein (EGFP) as the target protein, to achieve the fusion expression of the insect epidermal protein and EGFP. With chitin/chitosan materials as the stationary phase, the application potential of CPAP3 in protein purification was explored through binding experiments. The results showed that the fusion protein CPAP3-EGFP could bind to four different types of chitin/chitosan. Orthogonal experiment analysis optimized the reaction conditions as follows: β-chitin as the matrix and binding buffer at pH 6.5, under which the adsorption rate reached 70.44%. To further investigate the relationship between the number of domains and binding capacity, we conducted truncation treatment on CPAP3. The results indicated that the protein containing three domains had significantly higher binding capacity than the other two truncated groups, suggesting a positive correlation between the binding capacity of the protein tag and the number of domains. To effectively release the target EGFP bound to chitin, we introduced a TEV protease cleavage site between the target protein and the tag protein. Experimental results showed that after digestion with TEV protease, the protein sample exhibited a new band at 28 kDa, confirming that the fusion protein could be cleaved by TEV protease and the target protein was released into the supernatant. This study provides references for the application of insect chitin-binding proteins as affinity tags. 昆虫中存在多种几丁质结合蛋白。其中，含有ChtBD2几丁质结合域的CPAP3蛋白家族是昆虫表皮中含量最为丰富的蛋白质种类之一，本研究旨在以昆虫几丁质结合蛋白为标签蛋白，探讨其在蛋白纯化领域的应用潜力，同时探究CPAP3蛋白结构域数量与其结合能力的关系。利用家蚕CPAP3-A蛋白(简称CPAP3)作为标签蛋白，以绿色荧光蛋白EGFP为目标蛋白，构建融合表达体系实现二者融合表达。以几丁质/壳聚糖材料作为固定相，通过结合实验验证融合蛋白的结合能力。结果显示，融合蛋白CPAP3-EGFP与4种不同的几丁质/壳聚糖均能发生结合。正交实验表明最佳结合条件为β-几丁质、结合缓冲液pH值6.5，此时吸附率高达70.44%左右。为进一步探究结构域数量与结合能力的关系，对CPAP3蛋白进行了截短处理。结果表明，含有3个结构域的蛋白结合能力显著高于其他2个截短组，说明蛋白标签的结合能力与结构域数量呈正相关。为了有效释放结合在几丁质上的目标EGFP蛋白，在目的蛋白与标签蛋白之间引入了TEV蛋白酶切序列。实验结果显示，TEV蛋白酶酶切后，蛋白在28 kDa处出现了新的条带，证实融合蛋白能够被TEV酶切开，目标蛋白得以释放至溶液上清中。本研究为昆虫几丁质结合蛋白作为亲和标签的应用提供了参考。.
Objective Prokaryotic expression and purification of the Rift Valley fever virus (RVFV) nucleocapsid protein (NP) were performed to establish a basis for serum NP antibody detection and to produce specific polyclonal antibodies against NP. Methods The constructed pET28a-ZH501-NP prokaryotic expression plasmid was transformed into BL21(DE3) competent cells, and the expression of the recombinant protein NP was induced by isopropyl-β-D-thiogalactoside (IPTG). The recombinant protein NP was purified using HisTrapTM HP and mixed with Freund's complete and incomplete adjuvants to immunize New Zealand white rabbits. NP polyclonal antibodies were prepared, and their specificity was identified by indirect immunofluorescence (IFA) and Western Blotting. Results The prokaryotic expression plasmid was successfully constructed, and expression was successfully induced in BL21(DE3) competent cells. The recombinant protein NP was expressed in the form of inclusion bodies. Western Blotting and IFA assays indicated that the polyclonal antibody could specifically recognize RVFV NP. Conclusion The pure and effective expression of RVFV NP protein was achieved. The experimental conditions for the indirect ELISA using it as the coating antigen were explored. Simultaneously, specific polyclonal antibodies against RVFV NP were prepared, providing technical support for the establishment of a reliable rapid diagnostic technology for RVF and the research and evaluation of new RVFV vaccines.
Pseudorabies virus (PRV) glycoprotein E (gE) and glycoprotein B (gB) exhibit strong immunogenicity and serve as essential antigens for differentiating infected from vaccinated animals (DIVA). However, the expression and purification of gE and gB are often very difficult due to glycosylation. To evaluate the ability of poxvirus vectors to express glycosylated proteins, this study expressed PRV gE and gB via recombination vaccinia virus (VACV) vectors and assessed their biological activity. Genes encoding gE or gB were cloned and inserted into plasmid transfer vectors and integrated into VACV via homologous recombination. Recombinant viruses were purified through sucrose density gradient ultracentrifugation, followed by target protein purification via affinity chromatography. Rabbits were immunized with purified gE or gB proteins and boosted with the corresponding recombinant viruses (rVACV-gE or rVACV-gB). Post-boost antibody responses were significantly greater in both the gE and gB groups than in the control group. Antibodies obtained six weeks post-immunization showed binding affinity for wild-type PRV gE and gB. These results demonstrated that poxvirus vectors enable efficient, high-yield expression of complex viral glycoproteins.
The SCFSLY1 ubiquitin ligase complex is a key regulator of gibberellin signaling, mediating the degradation of DELLA proteins through targeted ubiquitination. To enable detailed biochemical and structural studies of this complex, efficient recombinant production of SLY1 and its adaptor ASK1 is essential. In this study, we optimized the co-expression and purification of the ASK1-SLY1 complex in Escherichia coli, focusing on molecular determinants that influence solubility, stability, and protein interaction. Hydrophobicity analyses and AlphaFold structural modeling of ASK1 and SLY1 identified prominent hydrophobic surfaces, particularly around helix α7 of SLY1, which are involved in DELLA binding and potentially driving aggregation during purification. Based on these insights, we adopted a co-expression strategy with an MBP tag fused at the N-terminal end of one partner, which significantly enhanced solubility and enabled successful isolation of the intact ASK1-SLY1 complex. Overall, this work presents an optimized protocol for recombinant production of the ASK1-SLY1 complex and provides structural rationale for overcoming key challenges in its expression and purification.
Nuclear Protein 1 (NUPR1) is a critical modulator of numerous cellular processes, including cell cycle regulation, apoptosis, and oncogenic transformation. Its structural characterization is indispensable for understanding its multifaceted biological functions and for its validation as a potential therapeutic target in diseases. Although foundational studies have reported on the expression and purification of NUPR1, detailed and scalable protocols for large-scale production, a prerequisite for advanced biophysical techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, have remained largely unarticulated. This study describes a successfully developed, optimized, and scalable protocol for the high-yield production and purification of recombinant human NUPR1. This optimized protocol consistently yielded 2-3 mg of NUPR1 per liter of M9 minimal medium with greater than 95% purity and high monodispersity, as confirmed by SEC-MALS analysis, making it highly suitable for demanding structural biology applications. Furthermore, efficient isotopic labeling strategies incorporating 15N and 13C were successfully implemented, enabling detailed multi-dimensional NMR spectroscopic analysis. Preliminary NMR data, including backbone resonance assignments, unequivocally confirmed the intrinsically disordered nature of NUPR1. Secondary structure propensity analysis derived from these assignments indicated a characteristically low propensity for the formation of stable canonical secondary structures. This robust, well-characterized, and scalable expression and purification pipeline provides an essential and solid foundation for future detailed structural, dynamic, and functional investigations of NUPR1, which are crucial for dissecting its complex roles in cellular physiology and disease pathogenesis.
Mycoplasma pneumoniae (M. pneumoniae) is a major pathogen causing community-acquired pneumonia in children. Its pathogenic process relies on the adherence to and colonization of host respiratory epithelial cells. P1 protein is the primary adhesin of M. pneumoniae, directly mediating its binding to host cells. To explore the interaction mechanism between P1 recombinant protein and host cells, we conducted protein expression and purification, glutathione S-transferase (GST) pull-down assay, and transcriptome sequencing. The rP1-GST fusion protein was expressed under confirmed induction conditions (16 °C, 0.1 mM IPTG). GST pull-down assay identified 22 differentially expressed membrane proteins in the rP1-GST group, among which annexin A2 (ANXA2) and C-C chemokine receptor type 5 (CCR5) were significantly altered and interact with P1 adhesin. Both ANXA2 and CCR5 possessed multiple functions including protein binding, receptor activity and signal sensor activity. Transcriptome analysis indicated that differentially expressed genes from rP1-A549 cell interaction were significantly enriched in multiple Gene Ontology (GO) terms and KEGG pathways. These results suggest that ANXA2 and CCR5 may serve as potential binding partners of P1 adhesin. P1 adhesin may regulate host genes involved in mitochondria and energy metabolism. These findings provide clues for understanding the adhesion and pathogenesis of M. pneumoniae.
The Hepatitis C virus (HCV) core protein is crucial for viral assembly and pathogenesis; however, structural studies have been hampered by its instability and historical reliance on denaturing purification methods. Although a recent non-denaturing purification method for an E. coli-expressed HCV core 1-164 protein enabled initial characterization, the purified protein exhibited poor long-term stability owing to spontaneous proteolytic cleavage. This study aimed to identify the cleavage sites responsible for this degradation and to engineer a more stable variant. N-terminal sequencing of the degraded fragments revealed two major cleavage sites between the residues Tyr35-Leu36 and Arg47-Thr48. On the basis of these findings, we designed mutants with alanine substitutions at these sites. The combined Pro1/2 (L36A/L37A/R47A/T48A/T49A/R50A), was expressed and purified. Compared to the wild-type protein, the Pro1/2 mutant demonstrated a markedly higher yield during purification and significantly improved stability during storage at 4 °C, retaining approximately 60% of the intact protein after five days versus 20% for the wild-type. Importantly, the mutations did not alter the elution profile of the protein by size-exclusion chromatography, suggesting that the overall structure was preserved, and in silico epitope prediction indicated a minimal impact on antigenicity. The enhanced stability and yield of this Pro1/2 mutant provides a robust platform for future high-resolution structural analyses of the HCV core protein and supports its potential use in the development of therapeutics and vaccines.
Brown seaweeds of the genus Sargassum have long been used in East Asia as traditional remedies for inflammatory conditions, bacterial infections, and cancer. Sargassum thunbergii, a non-edible and underutilized species, is rich in bioactive fucoidan. Chito-oligosaccharides (COS), derived from marine chitin, have also been traditionally associated with intestinal health and inflammation modulation. However, the ethnopharmacological potential of combining S. thunbergii fucoidan (STF) and COS in intestinal inflammatory conditions remains unexplored. This study aimed to evaluate the combined protective effects of STF and COS on intestinal inflammation and barrier dysfunction, and to elucidate their complementary pharmacological actions using in vitro and in vivo models of dextran sulfate sodium (DSS)-induced intestinal injury. DSS-induced Caco-2 cell monolayers were used to assess cytotoxicity, inflammatory mediator production, epithelial barrier integrity, and tight junction protein expression. A DSS-induced zebrafish larval model was employed to evaluate in vivo anti-inflammatory activity, nitric oxide (NO) production, cell death, and mucus homeostasis. STF and COS were tested individually and in combination at different ratios (1:2; 1:1; 2:1), with sulfasalazine and dexamethasone used as reference drugs. Both STF and COS individually attenuated DSS-induced inflammatory responses by reducing the production of IL-6, IL-8, TNF-α, and PGE2, and partially restoring the expression of tight junction proteins ZO-1 and occludin. Notably, the STF-COS combination at a 2:1 ratio produced greater protective effects than either treatment alone, resulting in a more effective suppression of inflammatory mediators and a marked improvement in epithelial barrier integrity, comparable to conventional anti-inflammatory drugs. In zebrafish larvae, the STF-COS mixture significantly reduced DSS-induced NO overproduction and cell death, while restoring intestinal mucus homeostasis. The enhanced protective effects of the STF-COS combination are likely attributable to their complementary pharmacological properties, with sulfated fucoidan mediating anti-inflammatory activity and low-molecular-weight COS enhancing bioavailability and epithelial repair. These findings provide experimental evidence supporting the ethnopharmacological potential of combined marine-derived polysaccharides as natural therapeutic candidates for intestinal inflammatory disorders.
The heterodimeric human sweet taste receptor TAS1R2/TAS1R3 is a class C G-protein-coupled receptor responsible for detecting a wide range of sweet-tasting compounds. The Venus flytrap domain of hTAS1R2 (hTAS1R2-VFT) constitutes the main binding site for natural sugars and some noncaloric sweeteners, including sucralose, neotame, and acesulfame-K. However, its biophysical characterization has been limited by difficulties in producing soluble and functional protein, with most strategies relying on refolding from inclusions bodies. In this study, we report the successful expression of soluble, folded, and functional hTAS1R2-VFT using Escherichia coli and a N-terminal small ubiquitin-like modifier (SUMO) fusion protein, avoiding protein refolding from inclusion bodies. A two-step purification process (immobilized metal ion affinity chromatography followed by preparative gel filtration) yielded approximately 0.42 mg of pure protein per liter of culture. Circular dichroism spectroscopy and size-exclusion chromatography coupled with multiangle static light scattering analysis confirmed proper folding and monomeric behavior of SUMO-hTAS1R2-VFT. Functional characterization using intrinsic tryptophan fluorescence revealed that SUMO-hTAS1R2-VFT bound sweet-tasting compounds with affinities consistent with their physiological relevance. Neotame exhibited the highest affinity (Kd ≈ 2 μM), followed by acesulfame-K (Kd ≈ 116 μM) and sucralose (Kd ≈ 282 μM), while sucrose showed weak binding in the millimolar range. These affinities were in agreement with sweetness potency and comparable to those reported for the full-length TAS1R2 subunit. This work provides the first demonstration of soluble expression of functional hTAS1R2-VFT in bacteria, by adding a solubility tag, offering a robust and scalable platform for studying sweetener-receptor interactions and facilitating the rational design of novel sweeteners.
Zingiber zerumbet (L.) Roscoe ex Sm., native to tropical and subtropical Asia, has long been used to treat gastrointestinal disorders, allergies, asthma, and skin diseases. Zerumbone, its principal bioactive compound, has attracted interest for its anti-inflammatory, anticancer, antioxidant, antidiabetic, and antihyperlipidemic properties. Conventional therapies for atopic dermatitis (AD) often provide only transient relief and may cause adverse effects. Although modern immunomodulators show strong efficacy, their high-cost limits accessibility. Thus, identifying natural compounds with potent efficacy and minimal side effects is crucial. This study examined the therapeutic potential and mechanisms of zerumbone in AD-like inflammation. An in vitro model using TNF-α/IFN-γ (TI)-stimulated HaCaT keratinocytes was employed to evaluate inflammatory signaling and cytokine expression. In vivo, AD-like symptoms were induced in BALB/c mice using 2,4-dinitrochlorobenzene (DNCB). Pro-inflammatory cytokines and chemokines were quantified by RT-qPCR, while histopathological and physiological assessments were performed to evaluate skin integrity and inflammation. Zerumbone inhibited MAPK/NF-κB, STAT, and Akt phosphorylation in TI-stimulated HaCaT cells and suppressed pro-inflammatory cytokine and chemokine mRNA levels. It also restored filaggrin, loricrin, and involucrin expression, essential for skin barrier function. In DNCB-induced AD mice, zerumbone reduced erythema, transepidermal water loss, ear thickness, and scratching while improving hydration. It also downregulated inflammatory cytokines and enhanced barrier protein expression. Zerumbone exerts potent anti-inflammatory and barrier-restorative activities in both cellular and animal models of atopic dermatitis, supporting its potential as a promising natural therapeutic candidate for AD and related inflammatory skin disorders.
In this study, adults and plerocercoids of S. mansoni were cultivated in vitro to systematically analyze the components of the excretory-secretory proteins (ESPs) of Spirometra mansoni. Afterwards, the differentially expressed proteins (DEPs) were identified and protein components were examined using the Data Independent Acquisition (DIA) mode. A total of 944 proteins were identified, including 580 plerocercoid-specific proteins, whereas no specific proteins were found in adults. Quantitative analysis revealed that 607 proteins were significantly differentially expressed, with 390 upregulated in the plerocercoid group, and 217 upregulated in the adult group. Gene Ontology functional annotation revealed that the upregulated proteins in the plerocercoid group were significantly enriched in functions such as nitrogen compound metabolism, proteasome core complexes, and ion binding. Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that the DEPs were strongly correlated with signal transduction, signal transportation, and catabolism pathways. Moreover, metabolic network analysis revealed that key pathways included the pentose phosphate pathway and glycolysis/gluconeogenesis. In addition, indirect ELISA revealed that immunization of mice with ESPs induced a Th1/Th2 mixed immune response, dominated by a Th1 response. Cytokine detection further verified that ESPs had good immunogenicity, and could activate both humoral and cellular immune responses. This study revealed, for the first time, the differential expression profile of ESPs between adults and plerocercoids of S. mansoni. These findings offer a potential reference for the diagnosis and prevention of sparganosis. Première analyse des protéines d’excrétion-sécrétion de Spirometra mansoni par la méthode 4D-DIA. Afin d'analyser systématiquement les composants des protéines d'excrétion-sécrétion (PES) de Spirometra mansoni, les adultes et les plérocercoïdes de S. mansoni ont été cultivés in vitro. Les protéines différentiellement exprimées (PDE) ont ensuite été identifiées et leurs composants analysés par la méthode d'acquisition de données indépendante (DIA). Au total, 944 protéines ont été identifiées, dont 580 spécifiques aux plérocercoïdes, tandis qu'aucune protéine spécifique n'a été trouvée chez les adultes. L'analyse quantitative a révélé que 607 protéines étaient significativement différentiellement exprimées, dont 390 surexprimées chez les plérocercoïdes et 217 chez les adultes. L'annotation fonctionnelle Gene Ontology a révélé que les protéines surexprimées chez les plérocercoïdes étaient significativement enrichies en fonctions telles que le métabolisme des composés azotés, les complexes centraux du protéasome et la liaison aux ions. L'analyse d'enrichissement des voies métaboliques de la Kyoto Encyclopedia of Genes and Genomes (KEGG) a révélé une forte corrélation entre les protéines différentiellement exprimées (PDE) et les voies de transduction et de transport du signal, ainsi que les voies de catabolisme. De plus, l'analyse du réseau métabolique a mis en évidence l'implication des voies des pentoses phosphates et de la glycolyse/gluconéogenèse. Par ailleurs, un test ELISA indirect a montré que l'immunisation de souris avec des ESP induisait une réponse immunitaire mixte Th1/Th2, à prédominance Th1. La détection des cytokines a confirmé la bonne immunogénicité des ESP et leur capacité à activer les réponses immunitaires humorales et cellulaires. Cette étude révèle pour la première fois le profil d'expression différentielle des ESP entre les adultes et les plérocercoïdes de S. mansoni. Ces résultats offrent ainsi une piste potentielle pour le diagnostic et la prévention de la sparganose.
Endocervical adenocarcinoma comprises biologically heterogeneous sub-types classified as human papillomavirus (HPV)-associated and HPV-independent, practically based on p16 immunohistochemistry according to the 2020 World Health Organization (WHO) classification. This study explored the prognostic relevance of WHO-defined HPV-associated versus HPV-independent histologic classification and examined associations between primary treatment modality and clinical outcomes in locally advanced endocervical adenocarcinoma. We conducted a single-center retrospective cohort study including patients diagnosed with International Federation of Gynecology and Obstetrics (FIGO) 2018 stage IB3 to IVA endocervical adenocarcinoma treated between 2007 and 2024. All cases underwent expert slide review, and pre-treatment HPV testing was assessed when available. Patients were analyzed according to histologic sub-type and heterogeneous primary treatment approaches, including radical hysterectomy-based treatment with or without adjuvant therapy (n = 65) and definitive concurrent chemoradiation therapy-based treatment with or without completion hysterectomy (n = 37). Progression-free survival and overall survival were evaluated using Cox proportional hazards models. Among 102 patients, 53 had HPV-associated tumors and 49 had HPV-independent tumors, the latter predominantly gastric-type adenocarcinoma. HPV-independent tumors were associated with significantly worse progression-free survival and overall survival compared with HPV-associated tumors. Within the HPV-independent cohort, primary treatment modality was independently associated with both progression-free and overall survival, with radical hysterectomy-based treatment showing more favorable progression-free survival estimates compared with definitive chemoradiation therapy. A programmed death-ligand 1 combined positive score <1 was independently associated with overall survival. WHO-defined HPV-associated versus HPV-independent histologic classification was a strong prognostic marker in locally advanced endocervical adenocarcinoma. In exploratory analyses of HPV-independent tumors, associations between primary treatment modality and survival outcomes were observed; however, these findings should be interpreted with caution because of treatment selection bias and the retrospective study design. Further prospective studies are warranted to refine risk stratification and multi-disciplinary treatment strategies for this aggressive sub-group.
Fusobacterium nucleatum is a Gram-negative obligate anaerobic bacterium with trans-organ pathogenicity. It serves as a crucial pathogen causing intracranial anaerobic infections and is closely associated with the occurrence of brain abscess. However, the mechanism by which Fusobacterium nucleatum crosses the blood-brain barrier and induces intracranial infection remains unclear. In this study, Fusobacterium nucleatum clinical strain isolated from the brain abscess was identified and characterized. Its growth characteristics and biofilm-forming capacity were compared with those of the ATCC 25586 standard strain. Furthermore, the adhesive and invasive capabilities of both strains towards oral epithelial cells and brain microvascular endothelial cells were assessed. An in vitro BBB model was constructed using a brain microvascular endothelial monolayer to examine the effects of bacterial infection on the expression of tight junction proteins (occludin, Claudin-5, ZO-1). Finally, antimicrobial susceptibility testing combined with proteomic techniques was employed to analyze the antibiotic resistance profiles and differentially expressed proteins of the strains. One clinical strain was successfully isolated and identified from specimens of 7 brain abscess patients. Compared to the standard strain, the clinical isolate demonstrated a slower growth rate, reduced biofilm formation, and diminished adhesion and invasion capabilities against both oral epithelial cells and brain microvascular endothelial cells. The two strains exhibited significantly distinct regulatory patterns on the tight junction protein expression in brain microvascular endothelial cells. Proteomic analysis revealed extensive protein reprogramming in the clinical strain, characterized by the upregulation of proteins involved in metabolic pathways and immune evasion. Altered antimicrobial susceptibility in the clinical strain correlated with differential expression of proteins such as ribosomal components and efflux pump proteins. Fusobacterium nucleatum can specifically adhere to and invade brain microvascular endothelial cells, disrupting their tight junctions. Through proteomic reprogramming, Fusobacterium nucleatum enhances its metabolic adaptation and immune evasion capabilities to adapt to changes in the intracranial microenvironment and establish persistent infection, thereby achieving a strategic shift from barrier penetration to intracranial colonization.
Pulmonary fibrosis (PF) is a progressive interstitial lung disease characterized by alveolar epithelial injury, inflammation, and excessive extracellular matrix deposition, yet current therapeutic options remain limited. Panax ginseng C.A. Meyer, a renowned qi-tonifying herb in traditional Chinese medicine, has long been used to enhance spleen and lung function by replenishing qi. However, the mechanism of action of its primary active component, ginseng stem and leaf saponins (GSLS), in pulmonary fibrosis remains incompletely understood. This study aims to elucidate the protective role of GSLS against pulmonary fibrosis by investigating how GSLS regulates mitochondrial transcription factor A (TFAM)-mtDNA homeostasis and suppresses PANoptosis in alveolar epithelial cells. The major constituents of GSLS were identified using UHPLC-Q Exactive HFX. A BLM-induced mouse model of pulmonary fibrosis and an MLE-12-primary fibroblast co-culture system were established to evaluate the therapeutic effects of GSLS. Surface plasmon resonance (SPR) and cell thermal shift assays (CETSA) were performed to assess the direct interaction and thermal stability between GSLS and TFAM. Co-immunoprecipitation (Co-IP), RT-qPCR, and immunofluorescence were used to evaluate PANoptosome assembly, cytosolic double-stranded DNA (dsDNA) leakage, and fibrotic marker expression. GSLS was found to contain multiple active ginsenosides, including Rk2, CK, Rk3, and Rf. In vivo, GSLS markedly alleviated BLM-induced lung pathological injury, reduced collagen deposition, and decreased oxidative stress. In vitro, GSLS directly bound to TFAM and enhanced its thermal stability. RT-qPCR and immunofluorescence analyses further demonstrated that GSLS effectively suppressed abnormal cytosolic dsDNA leakage, reduced MLE-12 cell death, and ameliorated mitochondrial dysfunction. Moreover, GSLS inhibited the assembly of the PANoptosome complex, suppressed epithelial PANoptosis, and decreased the expression of fibrosis-related proteins. GSLS mitigates BLM-induced pulmonary fibrosis by stabilizing TFAM, maintaining mtDNA homeostasis, and suppressing PANoptosis. This clarifies the potential mechanism of GSLS therapy for PF.
This study established a method for extracting and purifying foxtail millet lipoxygenase (LOX) by combining ammonium sulfate precipitation and ion exchange chromatography, and investigated its enzymatic properties. The purified LOX showed high specific activity (448.82 ± 13.26 U/mg) and a molecular weight of 96.6 kDa. It achieved maximum catalytic activity at 30 °C and pH 7.0, with good stability within the pH range of 5.0-6.0. Circular dichroism and ultraviolet spectral analyses demonstrated that the heat-induced reduction in LOX enzymatic activity derived from alterations in the protein's spatial structure. The enzymatic reaction kinetics of LOX followed the Michaelis-Menten equation, characterized by a Km of 3.62 ± 0.06 mM and a Vmax of 20.97 ± 0.24 mmol L-1·min-1. Cu2+, Mn2+, Ca2+, chelators, sulfhydryl reagents, and three antioxidants (ascorbic acid, quercetin, rutin) inhibited LOX activity (P < 0.05). Fluorescence spectroscopy analysis demonstrated that the fluorescence quenching of LOX by quercetin and rutin conformed to a static quenching process. Combined molecular docking and thermodynamic analyses showed that quercetin interacted with LOX mainly through hydrophobic forces, while rutin bound via hydrogen bonds and van der Waals forces. This work lays a theoretical foundation for the functional study of LOX and its application in food processing, quality improvement, and preservation.
Staphylococcus nepalensis has recently been identified in clinical settings, raising questions regarding its potential role as an emerging opportunistic pathogen. However, no vaccine-based preventive strategy has yet been proposed against this bacterium. The objective of this study was to characterize a clinical isolate using PCR-based molecular identification and to design a rational multi-epitope vaccine candidate against S. nepalensis through an integrated immunoinformatics-driven reverse vaccinology approach. Species-level identification was performed by PCR amplification and sequencing of the 16S rRNA gene. Antigenic proteins lacking predicted allergenic or toxic properties were selected for epitope mapping. Predicted linear B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes were further evaluated for sequence conservancy across available strains to ensure broad-spectrum coverage. Conserved epitopes were assembled into a single construct incorporating β-defensin and a PADRE sequence to enhance immunogenic potential. Structural modeling and validation suggested acceptable stereochemical quality, with 94.8% of residues located in favored regions of the Ramachandran plot. Disulfide engineering was performed to improve structural stability of the vaccine construct. Protein-protein docking computationally predicted favorable binding interactions between the vaccine construct and human TLR2 and TLR4 receptors, with ClusPro docking scores of -1489.2 and -1198.1, respectively. Molecular dynamics simulations suggested dynamic stability of the complexes over a 100 ns trajectory. Immune simulation analyses indicated the potential activation of both humoral and cellular immune responses, including antibody production and cytokine secretion. Codon optimization and in silico cloning suggested possible expression feasibility in Escherichia coli. Collectively, this study presents a computational framework for multi-epitope vaccine design against S. nepalensis, and the findings require experimental validation to confirm biological efficacy and immunogenicity.
The heart 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB2) plays a critical role in glucose homeostasis and the pathophysiology of diabetes mellitus. However, the FBPase-2 domain remains less studied than its PFK-2 kinase counterpart. Therefore, in this study, PFKFB2 was overexpressed with molecular chaperones, and the kinetic properties of the FBPase-2 domain were studied. The purified PFKFB2 showed maximum activity at pH 7.5 in Tris-HCl buffer with Mg2+ as the optimal cofactor. The FBPase-2 domain showed a classical Michaelis-Menten curve with a Km of 0.73 μM and a Vmax of 0.164 U/mg for F2,6BP. kcat and kcat/Km were calculated to be 0.366 × 10-3 s-1 and 6.99 × 104 M-1 s-1, respectively. Circular Dichroism analyses of the purified enzyme showed 9.6% α-helix, 24.09% β-sheets, 28.9% turns, and 37.0% others as the secondary-structure composition of PFKFB2. The chaperone, GrpE, showed the lowest HADDOCK score (-267.2 ± 15.7) among all PFKFB2-chaperone docking complexes. Virtual screening identified citrate as a high-affinity modulator with a Ki of 14.9 μM. In silico mutagenesis indicated that single (Arg351Ala) and double (Arg351Ala and Tyr337Ala) mutations in Chain A of PFKFB2 (a homodimer protein) potentially destabilizes binding of citrate. In HepG2 and HEK293 cell lines, citrate decreased the activity of the FBPase-2 domain of PFKFB2, mRNA expression of PFKFB2, and glucose uptake and glucose production in a concentration-dependent manner. Thus, the characterisation of the FBPase-2 domain of PFKFB2 highlights its potential as a metabolic target for regulation of glucose homeostasis in diabetes mellitus.
Although gastric cancer (GC) exhibits significant genomic heterogeneity, the clinical implications of its immune microenvironment remain poorly understood. We retrospectively evaluated patients with GC who underwent gastrectomies between 2011 and 2014. The tumors were analyzed for Epstein-Barr virus (EBV), microsatellite instability-high (MSI-H), tumor-infiltrating lymphocytes (CD3), tumor-associated macrophages (CD68 and CD163), and programmed death-ligand 1 (PD-L1) expression. Tumors were classified using the modified The Cancer Genome Atlas scheme, and their clinical characteristics were compared. A total of 567 patients were classified into EBV (6%), MSI-H (10%), chromosomal instability-like (36%), and genomically stable-like (48%) subtypes. EBV tumors exhibited the highest PD-L1 expression (85%) and immune infiltration by CD3+ T cells (86%), CD68+ macrophages (58%), and CD163+ macrophages (40%). High CD68+ macrophage tumors were associated with advanced stages and worse 5-year disease-free survival (83% vs. 95%; P<0.001); however, this association was not independently significant after adjusting for the tumor-node-metastasis stage. PD-L1 expression did not significantly affect the survival outcomes. GC subtypes have distinct immune microenvironments that influence prognosis. Our findings highlight the prognostic and therapeutic potential of immune profiling in GC.