Protein expression and purification最新文献_第4页

Strategic truncation improves recombinant expression of buffalo Fibulin-5 (FBLN5) in Escherichia coli 策略截断提高水牛纤维蛋白-5 （FBLN5）在大肠杆菌中的重组表达

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-11-17 DOI: 10.1016/j.pep.2025.106858

Deviyani Mahajan , Neha Sarova , Jyoti Rustagi , Shwetali Prajapati , Jai Kumar Kaushik , Ashok Kumar Mohanty , Manoj Kumar Jena , Sudarshan Kumar

Background

Accurate early pregnancy detection is crucial for enhancing herd productivity in bovines. There is growing interest in identifying reliable protein biomarkers that can be used to develop rapid, sensitive, and cost-effective diagnostic tools. This study focused on buffalo fibulin-5 (FBLN5), an extracellular matrix glycoprotein involved in tissue remodeling and cell adhesion, and a potential early pregnancy biomarker.

Methods

In this study, we used the pET-32a(+) expression vector and three E. coli expression hosts. Heat-shock transformation was performed in chemically competent cells (CaCl₂ method), and the expression was optimized at 18 °C and 37 °C. Recombinant truncated FBLN5 was purified through immobilized metal affinity chromatography (IMAC) on nickel resin and subsequently characterized by SDS-PAGE and Western blotting.

Results

Optimization of various expression conditions, such as temperature, induction, incubation time, and additives, did not result in any detectable expression of full-length FBLN5. Therefore, a truncated version of FBLN5 was designed based on the predicted antigenic regions to increase its immunogenicity and simplify its recombinant expression. Truncated FBLN5 (Trx-tagged and 6 × His-tagged) expressed well in BL21Codon Plus (DE3)-RIL and Lemo21(DE3) at 18 °C with 1 mM IPTG induction. Furthermore, we successfully purified soluble truncated FBLN5 and confirmed its molecular weight (∼36 kDa) and identity using western blotting with enhanced chemiluminescence (ECL).

Conclusion

Truncation of full-length FBLN5 resulted in successful cloning, expression, and purification of this otherwise difficult to express protein. This study provides a solid foundation for future immunological studies aimed to evaluate FBLN5 potential as diagnostic marker in early pregnancy detection in bovine species.

背景：准确的早期妊娠检测对提高牛群生产力至关重要。人们对鉴定可靠的蛋白质生物标志物越来越感兴趣，这些标志物可用于开发快速、敏感和具有成本效益的诊断工具。这项研究的重点是水牛纤维蛋白-5 (FBLN5)，这是一种参与组织重塑和细胞粘附的细胞外基质糖蛋白，也是一种潜在的早期妊娠生物标志物。方法：本研究采用pET-32a（+）表达载体和3个大肠杆菌表达宿主。热冲击转化为化学活性细胞（CaCl2法），在18°C和37°C条件下表达最佳。重组截断FBLN5通过镍树脂固定化金属亲和层析（IMAC）纯化，并通过SDS-PAGE和Western blotting对其进行表征。结果：优化各种表达条件，如温度、诱导、孵育时间、添加剂等，均未检测到FBLN5全长的表达。因此，我们根据预测的抗原区域设计了截断版FBLN5，以提高其免疫原性，简化其重组表达。截断的FBLN5 （trx标记和6×His-tagged）在18°C、1 mM IPTG诱导下在BL21Codon Plus (DE3)-RIL和Lemo21（DE3）中表达良好。此外，我们成功地纯化了可溶性截断的FBLN5，并使用增强化学发光（ECL）的western blotting证实了其分子量（~ 36 kDa）和身份。结论：截断全长FBLN5，成功克隆、表达和纯化了这种难以表达的蛋白。本研究为进一步开展FBLN5作为牛早期妊娠诊断标志物的免疫学研究奠定了基础。

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引用次数: 0

Isolation, purification and identification of major allergens Cor a 9 and Cor a 14 in hazelnuts 榛子中主要过敏原Cor a9和Cor a14的分离、纯化和鉴定

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-12-04 DOI: 10.1016/j.pep.2025.106866

Weichao Zhu , Huahong Yu , Ying Zhang , Qishu Luo , Min Song , Qin Geng , Lihua Zhou , Zhihua Wu

Hazelnut (Corylus avellana) is a widely consumed nut with potent allergenic potential, and the isolation and purification of native hazelnut allergens are critical for the effective prevention and management of hazelnut allergy. In this study, two major allergens Cor a 9 and Cor a 14 were purified from hazelnut kernels via a sequential protocol involving pulverization, defatting, protein extraction, ammonium sulfate precipitation, dialysis desalting, and anion-exchange chromatography. Results demonstrated successful purification of the target allergens with high purity and intact conformational structure, which exhibited specific immunoreactivity with sera from hazelnut-allergic patients. A single preparation run yielded over 5 mg of Cor a 14 (purity >95 %) and more than 1 mg of Cor a 9 (purity >85 %). The established protocol is simple, requires minimal equipment, and offers high efficiency, laying a preliminary foundation for hazelnut allergen-related research. In conclusion, we successfully isolated and purified two major hazelnut allergenic proteins Cor a 9 and Cor a 14, providing a valuable material basis for investigating the sensitization mechanism of hazelnut allergy and developing diagnostic tools or hypoallergenic products.

榛子是一种广泛食用的具有强致敏性的坚果，天然榛子过敏原的分离纯化是有效预防和管理榛子过敏的关键。在本研究中，通过粉碎、脱脂、蛋白质提取、硫酸铵沉淀、透析脱盐和阴离子交换色谱等顺序程序，从榛子仁中纯化了两个主要的过敏原Cor a 9和Cor a 14。结果表明，目标过敏原纯化成功，具有高纯度和完整的构象结构，对榛子过敏患者血清具有特异性免疫反应性。单次制备可产生5mg以上的Cor a14（纯度95%）和1mg以上的Cor a9（纯度85%）。所建立的方案简单、设备少、效率高，为榛子过敏原相关研究奠定了初步基础。综上所述，我们成功分离纯化了两个主要的榛子致敏蛋白Cor a 9和Cor a 14，为研究榛子过敏的致敏机制、开发诊断工具或低致敏产品提供了有价值的物质基础。

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引用次数: 0

Bench-top NMR of water signals: A non-destructive tool for biomacromolecule characterization 水信号的台式核磁共振：生物大分子表征的非破坏性工具。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-11-19 DOI: 10.1016/j.pep.2025.106855

Tomoto Ura , Taiji Oyama , Chiaki Nishimura

Bench-top NMR spectroscopy has emerged as an accessible, non-destructive tool for analyzing biomacromolecules in solution. This review focuses on how water proton NMR (wNMR) provides sensitive readouts of protein states in solution. We highlight representative studies on monoclonal antibodies, insulin, and vaccines under diverse stresses, where wNMR measurements consistently correlate with aggregation and stability outcomes. In parallel, we revisit the fundamental principles of wNMR, focusing on the molecular origins of water relaxation and the roles of hydration dynamics, proton exchange, and water confinement near protein surfaces. To illustrate these mechanisms, we present simple illustrative experiments using bench-top wNMR and ATR-FTIR spectroscopy with aqueous salts and polymers, revealing how non-protein solutes modulate hydrogen-bond networks and water dynamics. Finally, we discuss emerging perspectives that extend the scope of bench-top NMR beyond protein formulations. Together, these developments position bench-top NMR not only as a practical tool for stability assessment but also as a promising framework for probing water-mediated phenomena across biopharmaceutical and soft-matter systems.

台式核磁共振波谱已成为分析溶液中生物大分子的一种方便、非破坏性的工具。本文综述了水质子核磁共振（wNMR）如何提供溶液中蛋白质状态的敏感读数。我们重点介绍了在不同压力下对单克隆抗体、胰岛素和疫苗的代表性研究，其中wNMR测量结果与聚集性和稳定性结果一致相关。同时，我们回顾了wNMR的基本原理，重点关注水弛豫的分子起源以及水合动力学、质子交换和蛋白质表面附近的水约束的作用。为了说明这些机制，我们使用wNMR和ATR-FTIR光谱对含水盐和聚合物进行了简单的说明性实验，揭示了非蛋白溶质如何调节氢键网络和水动力学。最后，我们讨论了将台式核磁共振范围扩展到蛋白质配方之外的新兴观点。总之，这些发展使台式核磁共振不仅作为稳定性评估的实用工具，而且作为探测生物制药和软物质系统中水介导现象的有前途的框架。

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引用次数: 0

Extraction and characterization of functional duckweed proteins using alkaline, enzymatic, and ultrasound-assisted techniques 利用碱性、酶和超声辅助技术提取和表征功能性浮萍蛋白。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-11-17 DOI: 10.1016/j.pep.2025.106860

Ayça Akyüz, Seda Ersus

The urgent need for sustainable, plant-based protein sources has brought aquatic crops like duckweed (Lemnaceae) into focus due to their high protein content, fast growth, and minimal resource requirements. This study aims to optimize and compare alkaline, enzyme-assisted, and ultrasound-assisted extraction techniques to produce high-yield, functional protein concentrates from Lemna minor. Key extraction parameters—pH, temperature, and solvent-to-solid ratio—were systematically optimized, identifying pH 9, 55 °C, and 5 mL/g as ideal conditions. Ultrasound treatment (100 % amplitude, 10 min) and Alcalase L enzyme application significantly enhanced protein yields. Ultrasound-assisted extraction proved most effective, increasing protein yield by more than fivefold compared to conventional alkaline methods, reaching 60.09 % protein content on a dry basis. The resulting protein concentrates displayed desirable functional properties, including excellent solubility (92.19 % at pH 9), foaming capacity (92.62 %), and emulsion activity, all of which are critical for food applications. Comprehensive structural and compositional analyses confirmed the presence of essential amino acids, high mineral content (notably calcium and phosphorus), and acceptable techno-functional behavior. SEM and FTIR analyses supported the structural integrity of the extracted proteins, while flowability assessments suggested suitability for powder-based formulations. This research demonstrates that ultrasound-assisted protein isolation from duckweed offers a scalable and efficient strategy for producing high-quality protein concentrates. These findings position duckweed as a promising, sustainable protein source with strong potential for incorporation into functional foods and novel plant-based formulations.

对可持续的植物性蛋白质来源的迫切需求使浮萍（Lemnaceae）等水生作物成为人们关注的焦点，因为它们蛋白质含量高，生长快，资源需求少。本研究旨在优化和比较碱性、酶辅助和超声辅助提取技术，以获得高产量、功能性的苦荬菜浓缩蛋白。系统优化了关键提取参数pH、温度和液固比，确定pH 9、55℃和5 mL/g为理想条件。超声处理（100%振幅，10分钟）和Alcalase L酶的应用显著提高了蛋白质产量。超声辅助提取被证明是最有效的，与传统的碱性方法相比，蛋白质产量提高了5倍以上，在干燥的基础上达到60.09%的蛋白质含量。所得到的蛋白质浓缩物显示出理想的功能特性，包括优异的溶解度（pH值9时为92.19%）、起泡能力（92.62%）和乳液活性，所有这些都是食品应用的关键。全面的结构和成分分析证实了必需氨基酸的存在，高矿物质含量（特别是钙和磷），以及可接受的技术功能行为。SEM和FTIR分析支持了提取蛋白质的结构完整性，而流动性评估表明粉末基配方的适用性。本研究表明，超声辅助从浮萍中分离蛋白质提供了一种可扩展和有效的生产高质量浓缩蛋白的策略。这些发现表明浮萍是一种有前途的、可持续的蛋白质来源，具有很强的潜力，可以被纳入功能性食品和新型植物性配方中。

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引用次数: 0

Comparative biophysical and functional analysis of TCZ-UFRJ, a potential biosimilar to Actemra Actemra潜在生物类似物TCZ-UFRJ的生物物理和功能比较分析

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-11-11 DOI: 10.1016/j.pep.2025.106841

Sanclayver Corrêa Araújo , Federico Francisco Marsili , Renata Guimarães Alvim , Katia Maria dos Santos Cabral , Heitor Affonso de Paula Neto , Yraima Cordeiro , Marcius da Silva Almeida , Leda dos Reis Castilho , Renato Sampaio Carvalho

Biosimilar antibodies have become increasingly significant in the pharmaceutical market, driven by the expiration of patents on many reference products. This study presents a comparative analysis between the originator tocilizumab, used to treat inflammatory diseases such as rheumatoid arthritis, and the biosimilar candidate TCZ-UFRJ, produced in HEK293 cells. The investigation focused on various aspects, including primary, secondary, and tertiary structures, intact mass analysis, glycosylation pattern, and functional testing using the IL-6-sensitive THP-1 cell line and ligand binding assay. LC-MS peptide mapping achieved amino acid full sequence coverage for both TCZ-UFRJ and Actemra, confirming the identity of the biosimilar candidate. Both antibodies exhibited a similar secondary structure with a characteristic beta-sheet spectrum, as determined by circular dichroism. The intrinsic tryptophan emission fluorescence profile confirmed a correctly folded tertiary structure for both mAbs. Intact mass analysis demonstrated similar molecular mass and glycoform profiles. Glycosylation analysis revealed similar glycosylation sites and the presence of major N-glycans in both Actemra and TCZ-UFRJ. Dynamic light scattering analysis indicated a monodisperse sample without the presence of oligomers for both. An LSPR ligand binding assay confirmed the interaction between TCZ-UFRJ and the IL-6 receptor, demonstrating specific binding affinity. Subsequent functional testing in the IL-6-sensitive THP-1 cell line validated the biological activity of TCZ-UFRJ, supporting its potential as a biosimilar candidate. These preliminary findings suggest that TCZ-UFRJ holds promise as a biosimilar candidate to Actemra, but further comprehensive studies, including non-clinical and clinical trials, are essential to establish its safety, efficacy, and overall similarity to the originator drug.

由于许多参考产品的专利到期，生物类似药抗体在制药市场上变得越来越重要。本研究提出了用于治疗炎症性疾病（如类风湿关节炎）的原始tocilizumab与HEK293细胞中产生的生物类似药候选TCZ-UFRJ之间的比较分析。研究集中在各个方面，包括一级、二级和三级结构、完整质量分析、糖基化模式，以及使用il -6敏感的THP-1细胞系和配体结合试验进行功能测试。LC-MS peptide mapping实现了TCZ-UFRJ和Actemra的氨基酸全序列覆盖，证实了该候选生物类似药的身份。两种抗体具有相似的二级结构，具有典型的β -片谱，由圆二色性确定。固有色氨酸发射荧光谱证实了这两种单克隆抗体的正确折叠三级结构。完整质量分析显示相似的分子质量和糖型谱。糖基化分析显示，在Actemra和tsz - ufrj中存在相似的糖基化位点和主要n -聚糖。动态光散射分析表明，样品是单分散的，没有低聚物的存在。LSPR配体结合实验证实了TCZ-UFRJ与IL-6受体之间的相互作用，显示出特异性的结合亲和力。随后在il -6敏感的THP-1细胞系中进行的功能测试验证了TCZ-UFRJ的生物活性，支持其作为候选生物类似药的潜力。这些初步研究结果表明，TCZ-UFRJ有望成为Actemra的生物类似药候选药物，但需要进一步的综合研究，包括非临床和临床试验，以确定其安全性、有效性和与原研药的总体相似性。

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引用次数: 0

L-arginine interferes with functional studies of amyloid proteins 精氨酸干扰淀粉样蛋白的功能研究。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-11-14 DOI: 10.1016/j.pep.2025.106854

H.P. Chethana, U. Rathan Kumar, Gunimala Chakraborty, Arshdeep Sidhu

Intrinsically disordered proteins/regions are abundant in cancer signalling pathways and neurodegenerative diseases like Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, etc. Purification of intrinsically disordered proteins can be challenging due to their sticky nature. For intrinsically disordered amyloid proteins, in-vitro aggregation studies are ideal experiments to study their liquid to solid transition. However, over-expression of these proteins in E. coli often results in insoluble protein fraction that ends up in cell-pellet as inclusion bodies, on lysis and centrifugation. Supplementing purification buffers with l-arginine is known to increase the solubility of proteins. For most of the structured proteins increasing solubility translates into a higher yield of functional proteins. However, for aggregation prone proteins associated with neurodegenerative diseases, like α-synuclein (Parkinson's disease), Aβ (Alzheimer's disease), fused in sarcoma (amyotrophic lateral sclerosis), etc. inclusion of l-arginine might interfere with aggregation studies. To test our hypothesis, we purified aggregation prone α-synuclein and fused in sarcoma protein in the presence and absence of l-arginine and studied their fibrillization. While recombinant FUS is difficult to prepare, purification of α-synuclein is well established but in all the protocols a significant amount of protein remains as insoluble fraction in the pellet. Inclusion of l-arginine increases the yield of protein purification by about 3 folds for both the proteins, but the resulting protein does not aggregate into fibrils thus showing that increased solubility of amyloid proteins (α-synuclein and fused in sarcoma) in the presence of l-arginine is not suitable for aggregation studies.

内在无序蛋白/区域在癌症信号通路和神经退行性疾病如帕金森病、肌萎缩侧索硬化症、阿尔茨海默病等中大量存在。由于其粘性，本质上无序的蛋白质的纯化可能具有挑战性。对于内在无序的淀粉样蛋白，体外聚集研究是研究其从液体到固体转变的理想实验。然而，这些蛋白在大肠杆菌中的过度表达通常会导致不溶性蛋白片段，最终在裂解和离心时作为包涵体进入细胞小球。已知用l-精氨酸补充纯化缓冲液可以增加蛋白质的溶解度。对大多数结构蛋白来说，溶解度的增加转化为功能蛋白的更高产量。然而，对于与神经退行性疾病相关的易聚集蛋白，如α-突触核蛋白（帕金森病）、Aβ（阿尔茨海默病）、肉瘤（肌萎缩性侧索硬化症）融合蛋白等，纳入l-精氨酸可能会干扰聚集研究。为了验证我们的假设，我们纯化了易于聚集的α-突触核蛋白，并在存在和不存在l-精氨酸的情况下融合在肉瘤蛋白中，并研究了它们的纤维化。虽然重组FUS很难制备，但α-突触核蛋白的纯化已经很好地建立了，但在所有的方案中，大量的蛋白质仍然是颗粒中的不溶部分。l-精氨酸的加入使两种蛋白的蛋白纯化率提高了约3倍，但所得蛋白不会聚集成原纤维，因此表明淀粉样蛋白（α-突触核蛋白和肉瘤融合蛋白）在l-精氨酸存在下的溶解度增加不适合聚集研究。

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引用次数: 0

Optimized production and coagulation activity of rhFVII in HEK293 cells for bleeding disorders 出血性疾病HEK293细胞rhFVII生成及凝血活性优化

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-12-03 DOI: 10.1016/j.pep.2025.106867

Sen Zou , Xiaoxiao Li , Jiajun Liu , Shuai Fan , Zhaoyong Yang

Hemophilia, a genetic disorder characterized by impaired blood clotting, necessitates innovative treatments. This study optimized the expression of recombinant human coagulation factor VII (rhFVII) in HEK293 cells to enhance its therapeutic efficacy. The human FVII gene was cloned into the pcDNA3.1 vector, and transient transfection was performed with an optimized DNA-to-PEI ratio (1:2) at 70 % cell density. Stable FVII-expressing cell lines were selected with G418, confirmed via RT-PCR, and adapted to suspension culture. rhFVII expression was analyzed using SDS-PAGE, Western blot, and ELISA, while its coagulation activity was evaluated by prothrombin time tests. Transient transfection yielded rhFVII concentrations of 10.22 μg/L and clotting activity of 232.46 %. Stable monoclonal HEK293 cells in suspension produced rhFVII at 11.22 mg/L with maximum coagulation activity of 563.17 % and high stability and safety. The findings underscore the successful optimization of rhFVII expression in HEK293 cells, paving the way for advancements in biopharmaceutical production and improved treatment options for bleeding disorders. Future research should focus on in vivo validation and further refinement of culture conditions to maximize protein yield and stability.

血友病是一种以凝血功能受损为特征的遗传性疾病，需要创新的治疗方法。本研究通过优化重组人凝血因子VII （rhFVII）在HEK293细胞中的表达，提高其治疗效果。将人FVII基因克隆到pcDNA3.1载体中，在70%细胞密度下，以优化后的dna - pei比（1:2）瞬时转染。用G418筛选稳定表达fvii的细胞系，通过RT-PCR确认，并适应悬浮培养。采用SDS-PAGE、Western blot和ELISA检测rhFVII的表达，采用凝血酶原时间检测rhFVII的凝血活性。瞬时转染的rhFVII浓度为10.22 pg/L，凝血活性为232.46%。稳定的单克隆HEK293悬浮细胞在11.22 mg/L的浓度下产生rhFVII，最大凝血活性为563.17%，稳定性和安全性高。这些发现强调了rhFVII在HEK293细胞中表达的成功优化，为生物制药的进步铺平了道路，并改善了出血性疾病的治疗选择。未来的研究应集中在体内验证和进一步完善培养条件，以最大限度地提高蛋白质产量和稳定性。

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引用次数: 0

Expression and functional characterization of recombinant soluble CD40 Ligand in prokaryotic systems 重组可溶性CD40配体在原核系统中的表达及功能表征

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-12-19 DOI: 10.1016/j.pep.2025.106874

Qi Xu , Xinying He , Jiale Bai , Hanlin Liu , Tao Hong , Suying Dang , Wei Zhang

Objective

To develop an optimized prokaryotic expression system for producing functional soluble CD40 ligand (sCD40L) and characterize its hemostatic activity.

Methods

The codon-optimized sCD40L gene (encoding residues 113–261) was synthesized and cloned into pET28a vector via BamHI/XhoI sites. After transformation into E. coli BL21(DE3), soluble expression was induced with 0.5 mM IPTG at 20 °C for 12 h. The recombinant protein was purified using nickel-affinity chromatography and analyzed by SDS-PAGE and Western blot. Biological activity was assessed through in vitro platelet aggregation and in vivo hemostasis assays.

Results

The pET28a-sCD40L expression vector was successfully constructed, and the protein purity reached more than 90 %. Western blot confirmed the expected ∼20 kDa sCD40L. Functionally, the recombinant sCD40L enhanced platelet aggregation in a dose-dependent manner, and significantly reduced secondary bleeding time in mice.

Conclusion

Our optimized prokaryotic system efficiently produces functional sCD40L without requiring eukaryotic post-translational modifications, providing a valuable tool for studying CD40L-mediated hemostasis and developing potential therapeutic applications.

目的建立一种功能可溶性CD40配体（sCD40L）的优化原核表达体系，并对其止血活性进行表征。方法合成密码子优化后的sCD40L基因（编码残基113 ~ 261），通过BamHI/XhoI位点克隆到pET28a载体上。转化大肠杆菌BL21（DE3）后，用0.5 mM IPTG在20℃下诱导可溶性表达12 h，镍亲和层析纯化重组蛋白，SDS-PAGE和Western blot分析重组蛋白。通过体外血小板聚集和体内止血试验评估其生物活性。结果成功构建了pET28a-sCD40L表达载体，蛋白纯度达到90%以上。Western blot证实了预期的~ 20 kDa sCD40L。功能上，重组sCD40L以剂量依赖性方式增强血小板聚集，并显著缩短小鼠继发性出血时间。结论优化后的原核系统无需真核细胞的翻译后修饰即可高效生产功能性sCD40L，为研究cd40l介导的止血和开发潜在的治疗应用提供了有价值的工具。

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引用次数: 0

Plasmid-transformed Bifidobacterium longum 105A secreting β-glucuronidase for prodrug conversion of SN-38 glucuronide 质粒转化的长双歧杆菌105A分泌β-葡萄糖醛酸酶，用于SN-38葡萄糖醛酸的前药转化

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-11-10 DOI: 10.1016/j.pep.2025.106844

Atsushi Saisho , Michiko Shimokawa , Rintaro Kubo , Yuri Enomoto , Shun'ichiro Taniguchi , Hiroaki Kobayashi

The development of tumor-selective prodrug activation strategies remains a major challenge in cancer pharmacotherapy.

In this study, we focused on SN-38 glucuronide (SN-38G), a highly hydrophilic prodrug with low membrane permeability that remains pharmacologically inactive unless hydrolyzed by β-glucuronidase. To enable localized activation of SN-38G within tumors, we engineered a recombinant Bifidobacterium longum 105A strain capable of secreting β-glucuronidase. The enzyme was efficiently secreted under anaerobic conditions and retained stable catalytic activity in mildly acidic and hypoxic environments that resemble the tumor microenvironment. In an MTT assay using CT26 colon carcinoma cells, co-treatment with β-glucuronidase and SN-38G induced marked growth inhibition, whereas SN-38G alone showed no cytotoxic effect. Furthermore, HPLC analysis of culture supernatants confirmed enzymatic conversion of SN-38G into the active metabolite SN-38.

Together, these results provide a proof-of-concept for a microbial-enhanced prodrug activation approach in which plasmid-driven expression in Bifidobacterium longum 105A enables targeted release of SN-38. This strategy may contribute to the development of tumor-localized drug production systems capable of selectively activating diverse anticancer prodrugs with distinct mechanisms of action.

肿瘤选择性前药激活策略的开发仍然是癌症药物治疗的主要挑战。在这项研究中，我们重点研究了SN-38葡萄糖醛酸盐（SN-38G），这是一种高度亲水的前药，具有低膜通透性，除非被β-葡萄糖醛酸酶水解，否则保持无药理活性。为了使SN-38G在肿瘤内的局部激活，我们设计了一株能够分泌β-葡萄糖醛酸酶的重组长双歧杆菌105A菌株。该酶在厌氧条件下有效分泌，在类似肿瘤微环境的弱酸性和低氧环境中保持稳定的催化活性。在使用CT26结肠癌细胞的MTT实验中，β-葡萄糖醛酸酶和SN-38G共同处理诱导了明显的生长抑制，而SN-38G单独处理没有细胞毒性作用。此外，培养上清的HPLC分析证实了SN-38G转化为活性代谢物SN-38。总之，这些结果为微生物增强的前药激活方法提供了概念证明，其中质粒驱动的长双歧杆菌105A表达能够靶向释放SN-38。这一策略可能有助于肿瘤局部药物生产系统的发展，该系统能够选择性地激活具有不同作用机制的多种抗癌前药。

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引用次数: 0

Partial purification and characterization acidophilic lipase from Bacillus cereus ALP E1 蜡样芽孢杆菌alpe1嗜酸脂肪酶的部分纯化及特性研究。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-03-01 Epub Date: 2025-12-09 DOI: 10.1016/j.pep.2025.106870

Nindy Novita Sari , Nurhasanah , Titin Haryati

The discovery of a new microbial lipase is crucial since its utilization has various industrial applications. Bacillus cereus is a strain that has emerged as a potential source of microbial lipase. Until now, the exploration of new lipases from Bacillus cereus remains limited. This study aims to obtain a lipase enzyme from Bacillus cereus ALP E1, as well as to purify and characterize the lipase produced by this strain. In this study, an extracellular acidophilic lipase from Bacillus cereus ALP E1 isolated from seawater at Panjang Port, Lampung, Indonesia was partially purified and characterized. Extracellular lipase was produced using submerged fermentation methods, and then partially purified with ammonium sulphate fractionation and dialysis. Partial purification successfully achieved a 12.31 purification fold with specific activity at 31083.14 U/mg. According to SDS-PAGE analysis, the purified lipase had a single dominant band estimated at 55 kDa. Lipase characterization revealed an optimal pH of 5, an optimal temperature of 40 °C, and the highest activity towards the substrate p-nitrophenyl stearate (C18). The lipase was activated by Ba²⁺ and deactivated by Mg²⁺. This lipase activity is enhanced by adding 1 % Tween 20, but it is deactivated by Tween 80. All tested organic solvents at 5 % concentration were able to activate the lipase with the optimized two-fold lipase activation using chloroform added. The results demonstrate that seawater is a promising source for discovering lipases with high activity. This complete characterization is important information and serves as a foundation for using lipase in appropriate industrial applications.

一种新的微生物脂肪酶的发现是至关重要的，因为它的利用具有多种工业用途。蜡样芽孢杆菌是一种菌株，已出现作为微生物脂肪酶的潜在来源。到目前为止，蜡样芽孢杆菌中新的脂肪酶的探索仍然有限。本研究旨在从蜡样芽孢杆菌ALP E1中获得一种脂肪酶，并对该菌株产生的脂肪酶进行纯化和鉴定。本研究对从印度尼西亚楠榜岛Panjang港海水中分离的蜡样芽孢杆菌ALP E1细胞外嗜酸脂肪酶进行了部分纯化和表征。采用深层发酵法生产胞外脂肪酶，经硫酸铵分馏和透析部分纯化。部分纯化获得了12.31倍的纯化倍数，比活性为31083.14 U/mg。根据SDS-PAGE分析，纯化的脂肪酶有一个单一的优势带，估计为55 kDa。脂肪酶的最适pH值为5，最适温度为40℃，对底物对硝基苯基硬脂酸酯（C18）的活性最高。脂肪酶被Ba2+激活，被Mg2+失活。添加1%的Tween 20可增强该脂肪酶的活性，但添加Tween 80可使其失活。所有测试的有机溶剂在5%浓度下都能激活脂肪酶，并添加氯仿对脂肪酶进行优化的两倍激活。结果表明，海水是发现高活性脂肪酶的理想来源。这个完整的表征是重要的信息，并作为在适当的工业应用中使用脂肪酶的基础。

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引用次数: 0