Protein expression and purification最新文献_第3页

Recombinant expression of crotoxin and comparison to native crotoxin as immunogens for anti-crotalid antivenoms 响尾蛇毒素的重组表达及与天然响尾蛇毒素作为抗响尾蛇抗体免疫原的比较。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-12-15 DOI: 10.1016/j.pep.2025.106871

Miguel Angel Mejía Sánchez , Samuel Cardoso Arenas , Ricardo Miranda Blancas , Herlinda Clement , Ligia Luz Corrales García , Adrián Marcelo Franco-Vásquez , Ivan Arenas , Alejandro Carbajal Saucedo , Gerardo Corzo

The crotoxin complex is identified as the primary lethal toxin in various species of rattlesnakes. It comprises two subunits: CtxA, a non-toxic protein, and CtxB, a minimally toxic enzymatic phospholipase A₂. These components exert neurotoxic effects by disrupting neuromuscular transmission, specifically by inhibiting the release of acetylcholine, thereby causing flaccid paralysis of the muscles. Due to crotoxin's high lethality, variable presence, and proportion within crotalid venoms, considerable interest has been directed toward its inhibition. In this context, the heterologous expression of a recombinant fusion protein designated as rCtxBA, which incorporates the amino acid sequences of both CtxA and CtxB subunits of crotoxin, has been documented. The rCtxBA protein was recombinantly expressed, recovered, and subsequently purified. New Zealand rabbits were immunized with the recombinants rCtxBA, rCTxB, and the native CtxBA. ELISA and Western blot analyses revealed that the anti-crotoxin antibodies specifically recognized venoms containing crotoxin by binding to both conformational and linear epitopes of crotoxin-like or related phospholipases. In vivo neutralization assays performed in mice demonstrated efficacy of the anti-rCtxBA antibodies against venoms from Crotalus mictlantecuhtli, C. scutulatus salvini, and C. tzabcan—species known to contain crotoxin—with ED₅₀ values of 2.8, 7.8, and 2.5 mg/3LD₅₀, respectively. Conversely, no neutralization effect was observed against the venom of C. molossus nigrescens, a species lacking crotoxin in its venom profile. These findings substantiate that native crotoxin, present in whole crotalid venoms, can be neutralized using recombinant crotalid antigens, thereby facilitating the development of effective neutralizing antibodies.

响尾蛇毒素复合物被确定为各种响尾蛇的主要致死毒素。它包括两个亚基：CtxA（一种无毒蛋白）和CtxB（一种低毒性酶促磷脂酶A2）。这些成分通过破坏神经肌肉传递，特别是通过抑制乙酰胆碱的释放来发挥神经毒性作用，从而引起肌肉的弛缓性麻痹。由于响尾蛇毒素的高致死率，变化的存在和比例在响尾蛇毒液中，相当大的兴趣已经指向其抑制。在这种情况下，重组融合蛋白rCtxBA的异源表达已被证实，该蛋白包含了响尾蛇毒素CtxA和CtxB亚基的氨基酸序列。重组表达rCtxBA蛋白，回收并纯化。用重组体rCtxBA、rCTxB和天然CtxBA免疫新西兰兔。ELISA和western blot分析显示，抗响尾蛇毒素抗体通过结合响尾蛇毒素样或相关磷脂酶的构象和线性表位特异性识别含有响尾蛇毒素的毒液。在小鼠体内进行的中和实验表明，抗rctxba抗体对含有Crotalus microlantecuhtli、C. scculatus salvini和C. tzabcan（已知含有Crotalus）的毒液有效，ED50值分别为2.8、7.8和2.5 mg/3LD50。相反，没有观察到对C. molossus nigrescens毒液的中和作用，这是一种在毒液中缺乏响尾蛇毒素的物种。这些发现证实，天然响尾蛇毒素存在于整个响尾蛇毒液中，可以使用重组响尾蛇抗原来中和，从而促进了有效中和抗体的发展。

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引用次数: 0

Comparative analysis of SUMO and his-tag fusion for soluble expression of the SARS-CoV-2 omicron RBD in E. coli SUMO与his-tag融合在大肠杆菌中可溶性表达SARS-CoV-2组粒RBD的比较分析

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-12-09 DOI: 10.1016/j.pep.2025.106869

Sadegh Zargan , Saba Mesdaghinia , Hasan Jalili , Bahareh Dabirmanesh , Khosro Khajeh

The soluble expression of the SARS-CoV-2 Receptor-Binding Domain (RBD) is fundamental for manufacturing protein-based countermeasures. Although E. coli is a favored host for rapid production, its utility is often constrained by the formation of insoluble aggregates, thereby limiting the supply for vaccine and diagnostic development. Given the persistent threat of emerging SARS-CoV-2 variants, the need for reliable, high-yield expression platforms to overcome this solubility challenge remains critical. This study aimed to achieve soluble expression of the Omicron RBD variant in E. coli using a computationally guided strategy. To support solubility-enhancing tag selection, a machine learning–based tool (TagSolver), trained on the UESolDS dataset, predicted a 61.98 % solubility probability for the SUMO–RBD. Two constructs were designed: one containing a SUMO tag and a His-tagged RBD as a control. Both were expressed in E. coli SHuffle® T7 cells, and solubility was assessed by SDS–PAGE after 8 h of induction at 22 °C. The SUMO–RBD was expressed in a soluble form, whereas the His-tagged RBD was insoluble. Following purification and SUMO protease cleavage, FTIR analysis indicated a native-like secondary structure enriched in β-sheets. These findings demonstrate, for the first time, the soluble expression of the SARS-CoV-2 Omicron RBD in E. coli and establish a refolding-free strategy for producing this antigen. Furthermore, the TagSolver predictor was introduced, which may accelerate the selection of solubility-enhancing tags.

SARS-CoV-2受体结合域（RBD）的可溶性表达是制造基于蛋白质的对策的基础。虽然大肠杆菌是快速生产的有利宿主，但它的效用往往受到不溶性聚集体形成的限制，从而限制了疫苗和诊断开发的供应。鉴于新出现的SARS-CoV-2变体的持续威胁，需要可靠、高产的表达平台来克服这种溶解度挑战仍然至关重要。本研究旨在利用计算引导策略在大肠杆菌中实现Omicron RBD变体的可溶性表达。为了支持增强溶解度的标签选择，基于机器学习的工具（TagSolver）在UESolDS数据集上进行了训练，预测SUMO-RBD的溶解度概率为61.98%。设计了两个结构：一个包含SUMO标签，一个包含his标记的RBD作为对照。这两种蛋白均在大肠杆菌SHuffle®T7细胞中表达，22°C诱导8 h后通过SDS-PAGE检测其溶解度。SUMO-RBD以可溶性形式表达，而his标记的RBD则不溶。经过纯化和SUMO蛋白酶裂解后，FTIR分析显示在β-片中富集了一个类似天然的二级结构。这些发现首次证实了SARS-CoV-2 Omicron RBD在大肠杆菌中的可溶性表达，并建立了一种生产该抗原的无折叠策略。此外，引入了TagSolver预测器，该预测器可以加速溶解度增强标签的选择。

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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 : 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

Generation of a polyclonal antibody that specifically recognizes the recombinant NS5A protein of bovine viral diarrhea virus 特异性识别牛病毒性腹泻病毒重组NS5A蛋白的多克隆抗体的制备。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-12-08 DOI: 10.1016/j.pep.2025.106868

Weijie Zhou, Yuting Wang, Shaobo Liang, Jingjin Hu, Feng Pang

Bovine viral diarrhea virus (BVDV) is an economically important pathogen for causing bovine viral diarrhea (BVD), with a significant detrimental impact on cattle production worldwide. While the BVDV NS5A protein is known to be essential for replication, its function remain incompletely characterized. This study entailed cloning the NS5A gene and subsequently generating the recombinant plasmid pET-28a-NS5A for expression in Escherichia coli. The purified and refolded recombinant NS5A protein was employed to immunize New Zealand white rabbits, resulting in the production of specific polyclonal antibodies. The antibody was then deployed to investigate the expression of the NS5A protein. Our findings confirmed the NS5A protein was successfully produced and purified in a prokaryotic system. The generated anti-NS5A polyclonal antibody in rabbits showed a robust titer of 1:819,200. The antibody specifically detected the NS5A protein expressed in both prokaryotic and eukaryotic systems. With its high specificity and titer, this antibody may serve as a valuable reagent for investigating NS5A's functions in BVDV pathogenesis. It also holds potential for application in developing diagnostic assays and novel control strategies against BVD.

牛病毒性腹泻病毒（Bovine viral diarrhea virus， BVDV）是引起牛病毒性腹泻（Bovine viral diarrhea， BVD）的一种经济上重要的病原体，对世界范围内的牛生产造成了重大的不利影响。虽然已知BVDV NS5A蛋白对复制至关重要，但其功能尚未完全确定。本研究通过克隆NS5A基因，生成重组质粒pET-28a-NS5A在大肠杆菌中表达。将纯化后的重组NS5A蛋白重新折叠免疫新西兰大白兔，产生特异性多克隆抗体。利用抗体检测NS5A蛋白的表达情况。我们的发现证实了NS5A蛋白在原核系统中被成功地产生和纯化。兔抗ns5a多克隆抗体的效价为1:819 200。该抗体特异性检测原核和真核系统中表达的NS5A蛋白。该抗体具有高特异性和高滴度，可作为研究NS5A在BVDV发病机制中功能的有价值试剂。它也具有潜在的应用在开发诊断分析和新的控制策略，以对抗BVD。

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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 : 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

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 : 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

Discovery and development of bispecific antibodies. 双特异性抗体的发现和发展。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-12-01 Epub Date: 2025-08-05 DOI: 10.1016/j.pep.2025.106787

Tetsuya Wakabayashi, Taichi Kuramochi

Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.

双特异性抗体（BsAbs）代表了基于抗体的治疗方法的重大突破，提供了同时参与两个不同靶点的独特能力。bsab有望发挥常规抗体药物无法达到的治疗效果。具体来说，它们正在被开发用于细胞间桥接、接近效应、双靶标抑制和依赖于两种抗原类型的细胞靶向。近年来，利用这种双重靶向能力，抗体药物的发现取得了进展，双特异性抗体已经在多个治疗领域推出。这些药物包括旨在增强t细胞杀伤活性和抑制生长因子的抗肿瘤药物、模拟凝血因子功能的药物和血管生成抑制剂。这篇综述强调了关键性的技术进步，这些技术进步已经克服了与bsab相关的制造挑战，使制药级产品的开发成为可能。我们使用emicizumab作为案例研究来说明这些发展。特别强调抗体工程技术和蛋白质纯化技术之间的关键协同作用，这在成功生产bsab中起着至关重要的作用。此外，我们还讨论了亲和色谱的最新创新，特别是耐碱性蛋白L树脂的开发，这些树脂显著改善了商业生产过程。我们研究了这些树脂独特的亲和行为及其对BsAb纯化的影响。这篇综合综述旨在为研究人员和行业专业人士提供对双特异性抗体在治疗应用中的现状和未来潜力的全面了解，突出了这一快速发展领域的技术挑战和创新解决方案。

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引用次数: 0

Solubilization and refolding of inclusion bodies by detergents. 洗涤剂对包裹体的增溶和再折叠作用。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-12-01 Epub Date: 2025-08-07 DOI: 10.1016/j.pep.2025.106791

Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto

Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.

重组蛋白在生物试剂和生物制药的开发中发挥着重要作用。在这里，我们将主要回顾重组蛋白在包涵体中表达时的重折叠，尽管增强可溶性表达的策略被描述为重折叠包涵体的替代方法。这些策略包括，但不限于，在细胞培养基中添加化学伴侣，修改细胞裂解缓冲液和使用提高溶解度的融合标签。另一种增强溶解度的方法是为膜蛋白生成脂质复合物，形成不溶性的无脂蛋白。在各种增溶和再折叠技术中，介绍了使用变性剂、碱性pH值和压力的增溶和再折叠技术，重点介绍了使用洗涤剂的增溶和再折叠技术，这是一种有效且经济的技术。综述了十二烷基硫酸钠、月桂酰谷氨酸、萨科齐和十六烷基三甲基铵的广泛应用。缓慢或逐步去除用于增溶的变性剂或离子洗涤剂似乎在成功的再折叠中发挥关键作用，通过在再折叠过程中保持蛋白质的溶解度。在碱性再折叠中，缓慢的pH调节也有助于维持蛋白质的溶解度。加压折纸时，少量盐酸胍辅助折纸。

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引用次数: 0

Analysis of the antibacterial and anti-inflammatory functions of bovine lactoferrin functional fragment expressed in Escherichia coli via engineering 大肠杆菌表达的牛乳铁蛋白功能片段抑菌抗炎功能的工程分析。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-29 DOI: 10.1016/j.pep.2025.106865

Hui Teng, Zizi Ding, Yujie Wang, Xueliang Liu, Xiangshan Tang, Xuewen Zhang

The bovine lactoferrin is a natural iron-binding glycoprotein that has attracted widespread attention due to its functions such as antibacterial, antiviral, anti-tumor, anti-inflammatory and immunomodulatory effects. In this study, the lactoferrin functional fragment (BlfFf) was expressed in Escherichia coli by genetic engineering expression. The expressed BlfFf mainly existed as inclusion bodies in the host cells. After collecting the inclusion bodies, the BlfFf purified polypeptide was obtained through dissolution, renaturation and further chromatography preparation. The antibacterial test of the purified BlfFf indicated that BlfFf did not exhibit antibacterial activity before pepsin hydrolysis. After hydrolysis by pepsin, BlfFf has obvious antibacterial properties against both Staphylococcus aureus and Escherichia coli represent Gram-positive and Gram-negative bacteria each. Cell experiments have demonstrated that the prepared BlfFf can significantly reduce the expression of inflammation-related genes in the cell model of LPS-stimulated macrophages (Raw264.7) with inflammation model, showing a significant inflammatory inhibitory effect. In Raw264.7 cells treated with BlfFf, the expression levels of various pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and iNOS were significantly decreased. Lower concentrations of BlfFf show a certain cell-promoting growth effect. This experiment demonstrated the multiple biological activities and functions of bacterial genetic engineering in expressing bovine lactoferrin peptides.

牛乳铁蛋白是一种天然的铁结合糖蛋白，因其具有抗菌、抗病毒、抗肿瘤、抗炎和免疫调节等功能而受到广泛关注。本研究利用基因工程技术在大肠杆菌中表达乳铁蛋白功能片段（BlfFf）。表达的BlfFf主要以包涵体形式存在于宿主细胞中。收集包涵体后，通过溶出、复性和进一步层析制备得到BlfFf纯化多肽。对纯化的BlfFf进行抑菌实验表明，在胃蛋白酶水解前BlfFf不表现出抑菌活性。经胃蛋白酶水解后，BlfFf对代表革兰氏阳性菌和革兰氏阴性菌的金黄色葡萄球菌和大肠杆菌均有明显的抑菌作用。细胞实验表明，制备的BlfFf可以显著降低lps刺激的巨噬细胞（Raw264.7）炎症模型中炎症相关基因的表达，显示出明显的炎症抑制作用。在经BlfFf处理的Raw264.7细胞中，各种促炎细胞因子IL-1β、TNF-α、IL-6和iNOS的表达水平均显著降低。低浓度BlfFf具有一定的促细胞生长作用。本实验验证了细菌基因工程表达牛乳铁蛋白多肽的多种生物学活性和功能。

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引用次数: 0

Characterization of a novel GH5 β-mannanase from Hahella sp. CR1 and its synergistic role in degradation of spent coffee grounds 来自Hahella sp. CR1的新型GH5 β-甘露聚糖酶的表征及其在废咖啡渣降解中的协同作用

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-29 DOI: 10.1016/j.pep.2025.106863

Melvin Chun Yun Tan, Kok Jun Liew, Faezah Mohd Salleh, Chun Shiong Chong

An efficient enzymatic degradation of plant biomass is essential for agricultural waste valorization and biofuel production. β-Mannanase is a key lignocellulolytic enzyme that targets mannan, a major component in plant cell walls. Hahella spp. are known to produce cellulolytic enzymes, but no β-mannanase was characterized from this genus up to date. In this study, a gene encoding for β-mannanase (HcrMan1) was identified and isolated from the genome of Hahella sp. CR1. HcrMan1 encodes a multidomain enzyme comprising a GH5 subfamily 8 catalytic domain, an immunoglobulin-like (Ig) linker domain, and a CBM2 substrate-binding domain. The presence of the rare Ig-like linker and a structurally distinct CBM2 makes its domain architecture unique compared to homologs. To investigate their contributions in the enzyme reaction, the wild-type HcrMan1 and two truncated variants (HcrMan1ΔCBM2 and HcrMan1ΔIgΔCBM2) were heterologously expressed and purified. Results revealed that the removal of Ig-like linker domain significantly affected protein solubility, whereas the deletion of CBM2 reduced the substrate-binding affinity but enhanced catalytic efficiency (K_cat/K_m) from 18.08 to 22.45. Truncated HcrMan1ΔCBM2 demonstrated a higher specific activity against locust bean gum (LBG) at optimal conditions (50 °C, pH 8.0) compared to its wild type. Furthermore, HcrMan1ΔCBM2 exhibited enhanced enzymatic performance when combined with the commercial cellulase CTec2 in saccharifying spent coffee grounds (SCG), achieving significant increase in reducing sugar release after alkali, ammonia and sonication pretreatment compared to CTec2 alone. These noteworthy findings highlight the compatibility and industrial relevance of engineered HcrMan1 in applications that involved with the utilization of lignocellulosic biomass.

有效的酶降解植物生物质对农业废弃物增值和生物燃料生产至关重要。β-甘露聚糖酶是一种针对甘露聚糖的关键木质纤维素水解酶，甘露聚糖是植物细胞壁的主要成分。已知Hahella属产生纤维素水解酶，但迄今尚未从该属中鉴定出β-甘露聚糖酶。本研究从Hahella sp. CR1基因组中鉴定并分离到一个编码β-甘露聚糖酶（HcrMan1）的基因。HcrMan1编码一种多结构域酶，包括GH5亚家族8催化结构域、免疫球蛋白样（Ig）连接结构域和CBM2底物结合结构域。罕见的类igg连接体和结构独特的CBM2的存在使其结构域结构与同源物相比是独特的。为了研究它们在酶反应中的作用，我们对野生型HcrMan1和两个截短变体（HcrMan1ΔCBM2和HcrMan1ΔIgΔCBM2）进行了异源表达和纯化。结果表明，去除Ig-like连接域显著影响蛋白质的溶解度，而CBM2的缺失降低了底物结合亲和力，但提高了催化效率（Kcat/Km），从18.08提高到22.45。截断的HcrMan1ΔCBM2在最佳条件（50°C, pH 8.0）下对刺槐豆胶（LBG）的比活性高于野生型。此外，HcrMan1ΔCBM2与商用纤维素酶CTec2联合使用时，在将废咖啡渣（SCG）糖化时表现出更强的酶促性能，与单独使用CTec2相比，碱、氨和超声预处理后还原糖的释放量显著增加。这些值得注意的发现突出了工程HcrMan1在涉及木质纤维素生物质利用的应用中的兼容性和工业相关性。

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