Protein expression and purification最新文献

Fine-tuning of leaky expression delivers soluble and functional recombinant alphainterferon in the cytoplasm of Escherichia coli 在大肠杆菌细胞质中，对泄漏表达的微调可提供可溶性和功能性的重组α干扰素

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-28 DOI: 10.1016/j.pep.2026.106895

Rodrigo Martins Bretas , Marcus Vinícius Alves da Silva , Sophie Yvette Leclercq , Diana Paola Gómez-Mendoza , Armando da Silva Cunha Jr. , Luciana Maria Silva Lopes

Alphainterferons are cytokines that became famous in the treatment of certain infections and tumors due to their antiviral, antiproliferative, and immunomodulatory activities. While the production of these small proteins in Escherichia coli is well established, high-level overexpression typically results in the formation of insoluble and inactive inclusion bodies that need to be processed through costly and lengthy steps. Here we describe an optimized, inducer-free expression system using E. coli BL21 (DE3) and a pET-based vector with the human IFN-α2a gene. By strategically transitioning the culture from high-temperature biomass accumulation (37 °C) to mild conditions (16 °C) during the early-midlog phase, we leveraged basal expression to favor folding over aggregation. This approach achieved a cytoplasmic solubility of up to 75 %. The protein was recovered using a streamlined one- or two-step ion-exchange chromatography protocol, resulting in maximum purified yields of 23.5 mg/L. Characterization tests confirmed the protein's identity, high purity, in vitro activity, and correct disulfide bonding pattern. This method provides a cost-effective alternative for the production of soluble alphainterferon by eliminating the need for chemical inducers and complex refolding processes.

α干扰素是一种细胞因子，因其抗病毒、抗增殖和免疫调节活性而在某些感染和肿瘤的治疗中闻名。虽然这些小蛋白在大肠杆菌中的生产已经建立，但高水平的过表达通常会导致形成不溶性和无活性的包涵体，需要经过昂贵和漫长的步骤来处理。本研究利用大肠杆菌BL21 （DE3）和基于pet的人IFN-α2a基因载体构建了优化的无诱导剂表达体系。通过战略性地将培养从高温生物量积累（37°C）过渡到温和条件（16°C），我们利用基础表达来支持折叠而不是聚集。这种方法实现了高达75%的细胞质溶解度。使用流线型的一步或两步离子交换色谱法回收蛋白质，最高纯化率为23.5 mg/L。鉴定试验证实了该蛋白的特性、高纯度、体外活性和正确的二硫键模式。该方法通过消除对化学诱导剂和复杂的再折叠过程的需要，为生产可溶性干扰素提供了一种具有成本效益的替代方法。

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引用次数: 0

Scalable production and characterization of recombinant pneumococcal histidine triad protein D in Escherichia coli for vaccine development 重组肺炎球菌组氨酸三联体蛋白D在大肠杆菌中的规模化生产和特性研究

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-13 DOI: 10.1016/j.pep.2026.106889

Prashant Jadhav , Hitesh Malviya , Jyoti Gokhale

Streptococcus pneumoniae causes 1.5 million deaths annually, primarily in low-income countries. Pneumococcal histidine triad protein D (PhtD), a conserved 92–110 kDa surface protein, is a promising serotype-independent vaccine candidate due to its roles in adherence, zinc homeostasis, and complement evasion. This study optimized scalable production of His-tagged PhtD in Escherichia coli BL21(DE3). The PhtD gene (serotype 2, strain D39) was cloned into pET30a(+), and expression was optimized using a full factorial design of experiments, achieving up to 19.8 mg/L soluble PhtD (>90 % purity) with 0.1 mM IPTG at OD₅₉₀ = 0.2 for 2 h. Lysozyme treatment (0.3 mg/mL) increased yield to 22 mg/L, a 120 % improvement over the control (10 mg/L). Purification involved sonication, ultrafiltration, and IMAC (80–100 mM imidazole wash, 200–300 mM elution), with 1 mM PMSF preventing ∼30 % yield loss from proteolysis. PhtD's identity (105–110 kDa), secondary structure (15.5 % helix, 30.4 % sheet), and thermal stability (Tm ∼102 °C) were confirmed by SDS-PAGE, Western blotting, FTIR, circular dichroism, NMR, and DSC. This optimized, statistically guided process delivers the highest reported yield of soluble full-length PhtD in cytoplasmic E. coli with documented native-like folding and exceptional stability (Tm = 102 °C), providing a robust and scalable platform for serotype-independent pneumococcal vaccine development.

肺炎链球菌每年造成150万人死亡，主要发生在低收入国家。肺炎球菌组氨酸三联体蛋白D （PhtD）是一种保守的92-110 kDa表面蛋白，由于其在粘附、锌稳态和补体逃逸中的作用，是一种有前途的血清型无关疫苗候选物。本研究优化了his标记的PhtD在大肠杆菌BL21（DE3）中的规模化生产。将PhtD基因（血清型2，菌株D39）克隆到pET30a（+）中，利用全因子实验设计优化表达，在OD590 = 0.2条件下，以0.1 mM IPTG处理2 h，可获得高达19.8 mg/L（纯度>； 90%）的可溶性PhtD。溶菌酶处理（0.3 mg/mL）使产量提高到22 mg/L，比对照（10 mg/L）提高了120%。纯化包括超声、超滤和IMAC （80-100 mM咪唑洗涤，200-300 mM洗脱），用1 mM PMSF防止蛋白水解产生约30%的收率损失。通过SDS-PAGE、Western blotting、FTIR、圆二色性、NMR和DSC证实了phd的特性（105-110 kDa）、二级结构（15.5%螺旋结构，30.4%片状结构）和热稳定性（Tm ~ 102°C）。这一优化的、统计指导的工艺在细胞质大肠杆菌中提供了最高的可溶性全长PhtD产量，具有文献记载的原生折叠和卓越的稳定性（Tm = 102°C），为不依赖血清型的肺炎球菌疫苗开发提供了一个强大和可扩展的平台。

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引用次数: 0

Detection of human PD-1 on T-cells using a fusion protein consisting of an anti-PD-1 single chain variable fragment linked to superfolder GFP 使用与超级文件夹gfp连接的抗pd-1单链可变片段组成的融合蛋白检测t细胞上的人pd-1。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2025-12-28 DOI: 10.1016/j.pep.2025.106876

Jong Shin , David Sanford , Alex A. Bohm , William Bachovchin , Peter A. Bullock

Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have dramatically improved approaches to cancer therapy. One such antibody is termed Nivolumab (trade name Opdivo) that selectively targets PD-1. We previously described a Nivolumab-based anti-PD-1 single chain variable fragment (scFv), termed the anti-PD-1 double mutant (dm) scFv, that tightly binds to PD-1 and blocks the interaction between PD-1 expressed on T-cells and PD-L1 on CHO cells. Extending these experiments, we now report a fluorescent reporter formed by the fusion of the anti-PD-1dm with superfolder (sf) GFP. Immunofluorescence and flow cytometry studies established that the anti-PD-1dm-sfGFP fusion protein can be used to detect PD-1 on the surface of PD-1 expressing Jurkat cells. Furthermore, using a humanized mouse strain that expresses human PD-1 and PD-L1, we report that when combined with an anti-CD3 antibody, the anti-PD-1dm-sfGFP can be used to identify PD-1 expressing T-cells within mouse tumor biopsies. Thus, we have developed a reagent for the quantitative determination of activated T-cells in tumor biopsies. Finally, we report that an additional fluorescent probe for the detection of PD-1 in vivo can be generated by linking, via maleimide chemistry, a derivative of the anti-PD-1dm to the near-infrared dye IR800.

破坏t细胞衍生的PD-1与免疫抑制配体（如PD-L1）之间相互作用的单克隆抗体极大地改善了癌症治疗方法(Tan等人（2016），Topalian (2015), Pardoll, (2012) Sharma和Allioson（2015）进行了综述；引用(1 - 4))。其中一种被称为Nivolumab（商品名Opdivo）的抗体选择性靶向PD-1。我们之前描述了一种基于nivolumab的抗PD-1单链可变片段（scFv），称为抗PD-1双突变体（dm） scFv，它与PD-1紧密结合，阻断t细胞上表达的PD-1和CHO细胞上表达的PD-L1之间的相互作用。在这些实验的基础上，我们现在报道了一个由抗pd -1dm与超级文件夹（sf） GFP融合形成的荧光报告蛋白。免疫荧光和流式细胞术研究证实，抗pd -1dm- sfgfp融合蛋白可用于检测表达PD-1的Jurkat细胞表面的PD-1。此外，利用表达人PD-1和PD-L1的人源化小鼠菌株，我们报道当与抗cd3抗体结合时，抗pd -1dm- sfgfp可用于识别小鼠肿瘤活检组织中表达PD-1的t细胞。因此，我们开发了一种用于肿瘤活检中活化t细胞定量测定的试剂。最后，我们报道，通过马来酰亚胺化学，将抗pd -1dm的衍生物与近红外染料IR800连接，可以产生用于体内检测PD-1的额外荧光探针。

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引用次数: 0

Removal of polysorbates degradation-related impurities by depth filter screening: A case study 深度过滤筛选去除聚山梨酯降解相关杂质：一个案例研究。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-30 DOI: 10.1016/j.pep.2026.106893

Chunxiao Xuan , Yangguang Xu , Xinang Cui, Tengfei Ma, Fengjuan Lv

Depth filtration (DF) is widely utilized as a clarification step in pharmaceutical manufacturing, concurrently removing process-related impurities such as host cell proteins (HCPs) and DNA. This study demonstrates DF's capability in eliminating polysorbates (PS)-degrading impurities (esterases and metals) that may compromise the long-term stability of biopharmaceuticals. By using mass spectrometry-based methods, screening experiments with various commercial depth filters revealed significant brand-dependent variations in impurity removal efficiency. Our findings provide valuable reference data for projects requiring HCP removal, particularly those targeting challenging esterase or metals contaminants.

深度过滤（DF）被广泛用于制药制造中的澄清步骤，同时去除与工艺相关的杂质，如宿主细胞蛋白（HCPs）和DNA。这项研究证明了DF在消除聚山梨酸酯（PS）降解杂质（酯酶和金属）方面的能力，这些杂质可能会损害生物制药的长期稳定性。通过基于质谱的方法，对各种商用深度过滤器进行筛选实验，发现杂质去除效率存在显著的品牌依赖性。我们的研究结果为需要去除HCP的项目提供了有价值的参考数据，特别是针对具有挑战性的酯酶或金属污染物的项目。

引用次数: 0

Bioinformatics analysis and preparation of polyclonal antibodies against the extracellular region of the Langya Virus attachment protein 狼牙病毒附着蛋白胞外区多克隆抗体的制备及生物信息学分析。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-22 DOI: 10.1016/j.pep.2026.106888

Shuxing Qiu , Xing Yin , Jinjiao He , Jiayou Zhang , Jinjing Guo , Yonghua Qi , Feifan Liu

The Langya virus attachment glycoprotein (LayV-G) is a major surface glycoprotein critical for host cell entry, membrane fusion, and the induction of neutralizing antibody production. A highly specific, reliable, and cost-effective anti-LayV-G antibody is urgently needed to comprehensively assess its multifunctional role in the viral life cycle. Here, we report the first successful production of LayV-G protein and corresponding polyclonal antibodies (pAbs) using a prokaryotic expression system. In this study, the extracellular domain of LayV-G containing strong antigenic epitopes was predicted using bioinformatics approaches and selected for recombinant antigen production, followed by codon optimization and synthesis of the candidate protein-encoding gene. We then constructed the truncated attachment glycoprotein (tG) with a C-terminal histidine tag (His-tag), (pET-22b-tG/his) recombinant plasmid and expressed the protein in E. coli BL21 (DE3) competent cells. SDS–PAGE and subsequent tandem mass spectrometry revealed the molecular weight of the tG/his recombinant protein to be ∼64 kDa (kD). Subsequently, the targeted protein was used to immunize New Zealand white rabbits and generate pAbs. The specificity and titer of the pAbs were determined by Western blotting, indirect fluorescence assay (IFA), and indirect ELISA. The antibody exhibited high specificity for tG/his in prokaryotic cells (Western blotting), and LayV-G expressed by the recombinant baculovirus system (IFA). In conclusion, the LayV-G protein and pAbs targeting it lays the foundation for further investigations into LayV-G structure and function, pathogenic LayV molecular mechanisms, and rapid LayV detection methods.

狼牙病毒附着糖蛋白（LayV-G）是一种主要的表面糖蛋白，对宿主细胞进入、膜融合和诱导中和抗体产生至关重要。迫切需要一种高特异性、可靠且具有成本效益的抗layv - g抗体来全面评估其在病毒生命周期中的多功能作用。在这里，我们报告了首次使用原核表达系统成功生产LayV-G蛋白和相应的多克隆抗体（pAbs）。在本研究中，利用生物信息学方法预测了LayV-G含有强抗原表位的细胞外结构域，并选择用于重组抗原生产，随后进行密码子优化和候选蛋白编码基因的合成。然后，我们用c端组氨酸标签（his -tag）构建了截短的附着糖蛋白（tG），（pET-22b-tG/his）重组质粒，并在大肠杆菌BL21 （DE3）感受态细胞中表达该蛋白。SDS-PAGE和随后的串联质谱分析显示tG/his重组蛋白的分子量为~ 64千道尔顿（kD）。随后，将目标蛋白免疫新西兰大白兔，生成pab抗体。采用Western blotting、间接荧光法（IFA）和间接酶联免疫吸附试验（ELISA）检测pAbs的特异性和效价。该抗体在原核细胞（Western blotting）和重组杆状病毒系统（IFA）表达的LayV-G中对tG/his具有高特异性。综上所述，LayV- g蛋白和靶向它的pab为进一步研究LayV- g的结构和功能、致病LayV分子机制以及LayV的快速检测方法奠定了基础。

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引用次数: 0

Robust aggregate separation by Sartobind Rapid A Protein A membrane sarbinding Rapid A Protein A膜的稳健聚集体分离。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-24 DOI: 10.1016/j.pep.2026.106890

Gaoya Yuan, Meng Qu, Yifeng Li

Protein A resins are the most widely used affinity media for product capture in antibody and Fc-fusion protein purification. However, they generally lack the capability to separate monomers from co-binding aggregates. Recently, Protein A membrane has emerged as a promising alternative to resin-based Protein A column. We previously demonstrated that Sartobind Rapid A, a Protein A membrane from Sartorius, shows much stronger aggregate separation capability than resin-based Protein A columns. In the current work, using an aggregate-rich culture harvest as feed material, we evaluated three mobile phase additives (i.e., NaCl, CaCl₂ and Arg·HCl) for their effects on monomer-aggregate separation by Sartobind Rapid A membrane. After identifying the conditions that provided the best resolution, we challenged Sartobind Rapid A membrane with four artificial samples containing different ratios (i.e., 15%, 30%, 45% and 60%) of aggregates. Analysis of the main elution peak portion by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) suggested that this Protein A membrane effectively removed most aggregates in all artificial samples regardless of the percentage of aggregates in them. Thus, the current study shows that aggregate separation by Sartobind Rapid A membrane is not only effective but also highly robust, which makes Protein A membrane a more attractive choice than Protein A column for product capture, especially for cases where the culture harvest contains high amounts of aggregates.

蛋白A树脂是抗体和fc融合蛋白纯化中最广泛使用的产物捕获亲和介质。然而，它们通常缺乏从共结合聚集体中分离单体的能力。近年来，蛋白A膜已成为树脂基蛋白A柱的一种很有前途的替代品。我们之前证明了Sartobind Rapid A，一种来自缝匠蝇的蛋白A膜，比树脂基蛋白A柱具有更强的聚集分离能力。在当前的工作中，我们以富含集合体的培养收获为原料，评估了三种流动相添加剂（即NaCl， CaCl2和Arg·HCl）对Sartobind Rapid A膜分离单体-集合体的影响。在确定了提供最佳分辨率的条件后，我们用含有不同比例（即15%，30%，45%和60%）聚集体的四种人工样品对Sartobind Rapid A膜进行了挑战。通过粒径排除色谱-高效液相色谱（SEC-HPLC）对主要洗脱峰部分的分析表明，无论聚集体的百分比如何，该蛋白A膜都能有效地去除所有人工样品中的大部分聚集体。因此，目前的研究表明，Sartobind Rapid A膜的聚集体分离不仅有效，而且高度稳健，这使得蛋白A膜比蛋白A柱更有吸引力的产品捕获选择，特别是在培养收获含有大量聚集体的情况下。

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引用次数: 0

Purification and characterization of the peroxiredoxin 5-thioredoxin 2 complex: unraveling the redox regeneration of peroxiredoxin 5 by thioredoxin 2 within the peroxiredoxin catalytic cycle 过氧还蛋白5-硫氧还蛋白2复合物的纯化与表征：揭示过氧还蛋白催化循环中硫氧还蛋白2对过氧还蛋白5的氧化还原再生

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-28 DOI: 10.1016/j.pep.2026.106896

Xiangwei Yang , Yifan Yang , Sizhe Gong , Li Wang , Qian Liu , Ruiqi Sun , Fu-Ming Lian

Peroxiredoxin (Prx) is a critical cysteine-based peroxidase that detoxifies reactive oxygen species to maintain cellular peroxide homeostasis and prevent oxidative stress. Peroxiredoxin 5 (Prx5 or PRDX5) is the unique atypical 2-cysteine Prx in humans, which is able to scavenge the peroxides through redox-active cysteines. However, the precise regeneration mechanism of Prx5 by thioredoxin (Trx) within the catalytic cycle of Prx is not yet well understood. Here, we developed a design strategy through disulfide bond crosslinking to demonstrate that both Cys151 and Cys47 of Prx5 are able to form intermolecular disulfide bonds with thioredoxin 2 (Trx2). Gel filtration was further performed to validate the formation of Prx5-Trx2 complex in solution. We presented a scheme for obtaining the purified Prx5-Trx2 complex, and elucidated that this complex is a heterotetramer with a 1:1 molar ratio of both proteins. Predicted structure model of Prx5-Trx2 complex revealed that each Trx2 protein engages exclusively with a single subunit of the Prx5 dimer, which is distinctly different from the interaction mode observed in yeast Ahp1-Trx2 complex. Collectively, these results provide novel mechanistic insights into the transient Prx5-Trx2 complex formation and the electron transfer from electron donor Trx2 to Prx5 during the catalytic redox regeneration step.

过氧化物还氧素（Prx）是一种重要的半胱氨酸过氧化物酶，它能解毒活性氧，维持细胞过氧化物稳态，防止氧化应激。过氧化物还氧蛋白5 （Prx5或PRDX5）是人类独特的非典型2-半胱氨酸Prx，它能够通过氧化还原活性半胱氨酸清除过氧化物。然而，在Prx的催化循环中，Prx5被硫氧还蛋白（Trx）再生的确切机制尚不清楚。在这里，我们开发了一种通过二硫键交联的设计策略，以证明Prx5的Cys151和Cys47都能够与硫氧还蛋白2 （Trx2）形成分子间二硫键。凝胶过滤进一步验证了溶液中Prx5-Trx2络合物的形成。我们提出了一种获得纯化Prx5-Trx2复合物的方案，并阐明了该复合物是两种蛋白质摩尔比为1:1的异四聚体。Prx5-Trx2复合体的预测结构模型显示，每个Trx2蛋白只与Prx5二聚体的一个亚基相互作用，这与酵母Ahp1-Trx2复合体的相互作用模式明显不同。总的来说，这些结果为Prx5-Trx2复合物的瞬态形成以及在催化氧化还原再生步骤中电子从电子供体Trx2转移到Prx5提供了新的机制见解。

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引用次数: 0

Additive-based protein stabilization for stress-prone and unstable proteins 基于添加剂的蛋白质稳定应力易感性和不稳定蛋白。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-10 DOI: 10.1016/j.pep.2026.106885

Tomoto Ura, Takumi Nakamura, Moe Iijima, Toya Yoshida, Kentaro Shiraki

Protein-stabilizing additives have traditionally been investigated under dilute solution conditions using simple monomeric proteins as model systems. While such studies have clarified fundamental additive–protein interactions, their direct applicability to practical research and industrial contexts remains limited. This review leverages insights from model systems to summarize additive-based stabilization strategies in two major directions. First, we address process-related stresses, focusing on high-concentration conditions and interfacial stress frequently encountered during protein handling. Second, we highlight strategies for protein classes that are challenging to stabilize or purify, including membrane proteins, viral spike proteins, and intrinsically disordered proteins. This review aims to encourage additive use when proteins are difficult to stabilize under challenging conditions.

传统上，用简单的单体蛋白质作为模型系统，在稀溶液条件下研究蛋白质稳定添加剂。虽然这些研究已经阐明了基本的添加剂-蛋白质相互作用，但它们对实际研究和工业背景的直接适用性仍然有限。本综述利用模型系统的见解，总结了两个主要方向的基于可加性的稳定策略。首先，我们解决了过程相关的应力，重点关注高浓度条件和蛋白质处理过程中经常遇到的界面应力。其次，我们重点介绍了难以稳定或纯化的蛋白质类的策略，包括膜蛋白、病毒刺突蛋白和内在无序蛋白。这篇综述的目的是鼓励添加剂的使用，当蛋白质在具有挑战性的条件下难以稳定。

引用次数: 0

Recombinant expression and purification of TANB77-derived pilin-like proteins 935 and 938 tanb77衍生的蛋白935和938的重组表达和纯化。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-02-01 DOI: 10.1016/j.pep.2026.106898

Tham Truong Phuong Tran, Linh Thanh Nguyen, Byeong-Gu Han, Min-Jung Lee, Byoung Choul Kim

Increasing evidence highlights the gut microbiota as a source of diagnostic biomarkers and a key modulator of anti-tumor immunity and cancer immunotherapy responses. Pilin-like proteins from the uncultured TANB77 clade have shown potential to enhance immune checkpoint blockade therapy, yet protocols for their recombinant production have not been established. Here, we present the first optimized method for the expression and purification of two TANB77-derived pilin-like proteins, 935 and 938, using the Escherichia coli (E. coli) BL21(DE3)/pET system. The workflow integrates optimized expression conditions, two-step purification by Ni-NTA affinity and size-exclusion chromatography (SEC), and endotoxin removal for downstream applications. We reveal significant differences in the expression profiles and biophysical properties of the two proteins: protein 935 was expressed at high levels (∼2.5 mg/100 mL) and formed stable oligomers. In contrast, protein 938 exhibited >30-fold lower yields (∼0.08 mg/100 mL) and significant fragmentation, indicating intrinsic instability within this expression system. This study provides the first systematic framework for producing TANB77 pilin-like proteins, facilitating future exploration of their biochemical properties and therapeutic potential in cancer immunotherapy.

越来越多的证据表明，肠道微生物群是诊断生物标志物的来源，也是抗肿瘤免疫和癌症免疫治疗反应的关键调节剂。来自未培养的TANB77分支的pilin样蛋白已显示出增强免疫检查点阻断治疗的潜力，但其重组生产的方案尚未建立。本文采用大肠杆菌BL21(DE3)/pET系统，首次优化了tanb77衍生的两种蛋白935和938的表达和纯化方法。该流程集成了优化的表达条件，Ni-NTA亲和和大小排除色谱（SEC）两步纯化，以及下游应用的内毒素去除。我们揭示了两种蛋白在表达谱和生物物理特性上的显著差异：蛋白935在高水平表达（~ 2.5 mg/100 mL）并形成稳定的低聚物。相比之下，蛋白938的产率低了30倍（约0.08 mg/100 mL），且片段化明显，表明该表达系统具有内在的不稳定性。该研究为TANB77蛋白样蛋白的生成提供了第一个系统框架，有助于进一步探索其生化特性和在癌症免疫治疗中的治疗潜力。

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引用次数: 0

Hyper-expression of functional human basic fibroblast growth factor 2 in Escherichia coli 功能性人碱性成纤维细胞生长因子2在大肠杆菌中的高表达。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2026-05-01 Epub Date: 2026-01-28 DOI: 10.1016/j.pep.2026.106894

Tamilvendan Manavalan , Jiawu Bi , Khongsay Naulchan , Nguyen Thi Bich Van , Xin Yi Chua , Ryan Yow , Sook Yee Lee , Lamony Jian Ming Chew , Christoph Ottenheim , Shigeki Sugii , Manikandan Lakshmanan , Weibiao Zhou , Prakash Arumugam , Fong Tian Wong , Melanie Weingarten , Ee Lui Ang

The human basic fibroblast growth factor 2 (hbFGF2) is a highly conserved protein that is crucial for cell proliferation, differentiation, and migration. Commercial hbFGF2 is expensive and contributes significantly to the cost of cultivated meat (CM) media, affecting the scalability of the production process. This study aims to reduce media costs by achieving a high yield of purified active hbFGF2 from Escherichia coli T7 Express. A codon-optimized hbFGF2 variant, hbFGF2-G3, was cloned into the pET-28a (+) plasmid and expressed in E. coli T7 Express. Cultivation parameters, including temperature and induction time, were optimized in both shake flasks and batch bioreactors. Optimized conditions yielded a titer of 1.3 g/L in shake flasks at 20 °C and an impressive 2.95 g/L in a batch bioreactor, quantified by densitometry using an in-house purified hbFGF2-G3 as the standard. The protein was subsequently purified using His-trap affinity and size exclusion chromatography. The functional activity of the purified hbFGF2-G3 was validated through FGF/FGFR activation assays in HepG2 cell lines and cell proliferation studies in Ph9F-1x fish cell lines. Our findings demonstrate a cost-effective method for producing active hbFGF2-G3, which has significant implications for the economic viability of CM production.

人碱性成纤维细胞生长因子2 （hbFGF2）是一种高度保守的蛋白，对细胞增殖、分化和迁移至关重要。商用hbbfgf2价格昂贵，对培养肉（CM）培养基的成本有很大贡献，影响了生产过程的可扩展性。本研究旨在通过从大肠杆菌T7 Express中获得高产量的纯化活性hbbfgf2来降低培养基成本。将密码子优化后的hbFGF2变体hbFGF2- g3克隆到pET-28a（+）质粒中，并在大肠杆菌T7 Express中表达。对摇瓶和间歇式生物反应器中温度、诱导时间等培养参数进行了优化。优化后的条件在20°C的摇瓶中滴度为1.3 g/L，在间歇式生物反应器中滴度为2.95 g/L，使用内部纯化的hbFGF2-G3作为标准进行密度测定。随后用His-trap亲和层析和大小排斥层析纯化该蛋白。通过HepG2细胞系的FGF/FGFR激活试验和Ph9F-1x鱼细胞系的细胞增殖研究，验证了纯化的hbFGF2-G3的功能活性。我们的研究结果证明了一种具有成本效益的生产活性hbFGF2-G3的方法，这对CM生产的经济可行性具有重要意义。

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引用次数: 0