Protein expression and purification最新文献_第5页

A kaleidoscope of hosts: Expression systems of recombinant antibody reagents for immunological assays 宿主的万花筒：用于免疫检测的重组抗体试剂的表达系统。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-15 DOI: 10.1016/j.pep.2025.106857

Jia Xuan Yeoh , Yee Siew Choong , Theam Soon Lim

Monoclonal antibodies (mAbs) have been shown to be highly promising reagents used in immunological analysis of various diseases and other chronic conditions due to their specific targeting, potency, and stability. Advances in protein engineering and immunology have led to the development of recombinant monoclonal antibodies at a staggering pace. Full-length antibodies IgG are generally the preferred format, but the development of smaller formats like Fab, scFv, and sdAbs opened the floodgates for more variation. This allowed for a more flexible application of mAbs in immunological assays. A diverse set of expression systems have been used to express recombinant mAbs which includes bacterial, yeast, insect, mammalian cells, plant, cell-free and Leishmania each with distinct advantages and disadvantages. This review highlights that the selection of an optimal expression system and antibody format must be guided by the intended application, balancing yield, structural integrity, and cost. While no single host fulfils all criteria, continued advances in host engineering, synthetic design, and AI-driven optimization are expected to streamline recombinant antibody production and expand its applicability across therapeutic and diagnostic fields.

单克隆抗体（mab）由于其特异性靶向、效力和稳定性，已被证明是非常有前途的试剂，用于各种疾病和其他慢性疾病的免疫学分析。蛋白质工程和免疫学的进步导致重组单克隆抗体以惊人的速度发展。全长抗体IgG通常是首选格式，但较小格式的发展，如Fab， scFv和sabs结构域打开了更多变化的闸门。这使得单克隆抗体在免疫学分析中的应用更加灵活。多种表达系统已被用于表达重组单克隆抗体，包括细菌、酵母、昆虫、哺乳动物细胞、植物、无细胞和利什曼原虫，每种表达系统都有其独特的优点和缺点。这篇综述强调了最佳表达系统和抗体格式的选择必须以预期的应用、平衡产量、结构完整性和成本为指导。虽然没有单一宿主能够满足所有标准，但宿主工程、合成设计和人工智能驱动优化的持续进步有望简化重组抗体的生产，并扩大其在治疗和诊断领域的适用性。

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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 : 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

Amino acid chemistry and post-translational modifications underlying marine adhesive proteins: Biochemical insights for designing underwater adhesives 氨基酸化学和翻译后修饰潜在的海洋粘合剂蛋白质：设计水下粘合剂的生化见解。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-12 DOI: 10.1016/j.pep.2025.106851

Taehee Yoon , Hyung Joon Cha

Underwater adhesion remains a complex challenge due to the interference of water with traditional bonding mechanisms. Marine organisms, such as mussels, sandcastle worms, and barnacles, have evolved specialized adhesive proteins that overcome these limitations through functional amino acid motifs. This review highlights how post-translationally derived amino acid motifs, such as 3,4-dihydroxylphenylalanine (DOPA) and phosphoserine, drive interfacial adhesion and the curing process through interfacial binding, metal coordination, and crosslinking. Alongside these, canonical amino acids, such as cysteine, lysine, arginine, histidine, glycine, and proline, contribute to redox buffering, electrostatic interactions, structural flexibility, and fibrillar assembly, which are essential for adhesive structure. The adhesive strategies of mussels, sandcastle worms, and barnacles reflect diverse mechanisms: mussels utilize DOPA-rich proteins with redox-regulation; sandcastle worms employ electrostatically driven coacervation and ion-mediated curing; barnacles generate nanostructured networks stabilized by disulfide bonding and hydrophobic packing. This review presents a unified molecular framework that links amino acid chemistry, biochemical transformations, and structural integration in underwater adhesion. It further discusses synthetic and recombinant approaches that mimic these natural systems, including catechol-functionalized polymers, complex coacervate adhesives, and genetically engineered proteins. These biomimetic platforms demonstrate the potential of translating marine adhesion logic into robust, water-compatible materials for biomedical and industrial applications.

由于水对传统粘接机制的干扰，水下粘接仍然是一个复杂的挑战。海洋生物，如贻贝、沙堡蠕虫和藤壶，已经进化出专门的粘附蛋白，通过功能性氨基酸基序克服了这些限制。本文综述了翻译后衍生的氨基酸基序，如3,4-二羟基苯丙氨酸（DOPA）和磷酸丝氨酸，如何通过界面结合、金属配位和交联驱动界面粘附和固化过程。除此之外，典型氨基酸，如半胱氨酸、赖氨酸、精氨酸、组氨酸、甘氨酸和脯氨酸，有助于氧化还原缓冲、静电相互作用、结构灵活性和纤维组装，这些对粘合剂结构至关重要。贻贝、沙堡虫和藤壶的粘附策略反映了不同的机制：贻贝利用富含多巴的蛋白质进行氧化还原调控；沙堡蠕虫采用静电驱动凝聚和离子介导固化；藤壶产生纳米结构的网络稳定的二硫键和疏水填料。本文综述了水下粘附中氨基酸化学、生化转化和结构整合的统一分子框架。它进一步讨论了模拟这些自然系统的合成和重组方法，包括儿茶酚功能化聚合物，复杂凝聚粘合剂和基因工程蛋白。这些仿生平台展示了将海洋粘附逻辑转化为生物医学和工业应用中坚固的水兼容材料的潜力。

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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 : 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

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 : 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。

{"title":"Plasmid-transformed Bifidobacterium longum 105A secreting β-glucuronidase for prodrug conversion of SN-38 glucuronide","authors":"Atsushi Saisho , Michiko Shimokawa , Rintaro Kubo , Yuri Enomoto , Shun'ichiro Taniguchi , Hiroaki Kobayashi","doi":"10.1016/j.pep.2025.106844","DOIUrl":"10.1016/j.pep.2025.106844","url":null,"abstract":"<div><div>The development of tumor-selective prodrug activation strategies remains a major challenge in cancer pharmacotherapy.</div><div>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 <em>Bifidobacterium longum</em> 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.</div><div>Together, these results provide a proof-of-concept for a microbial-enhanced prodrug activation approach in which plasmid-driven expression in <em>Bifidobacterium longum</em> 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106844"},"PeriodicalIF":1.2,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145493274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression, purification and functional validation of a cancer-associated isoform of the HBx protein from human hepatitis B virus 人乙型肝炎病毒HBx蛋白癌症相关亚型的表达、纯化和功能验证

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-03 DOI: 10.1016/j.pep.2025.106842

Alexis Clavier , Santiago Gómez-Evain , Toshinobu Shida , Rubaba R. Abanti , Franziska Hammerstein , Pavel Kielkowski , Mila Leuthold , Anne K. Schütz

The human hepatitis B virus (HBV) causes hepatitis B, a liver infection that can be acute or chronic. HBV encodes four proteins, among which the X protein (HBx) plays a critical role in viral replication. During chronic HBV infection, in which the viral DNA is integrated into the host genome, the HBx_1-120 isoform, comprising the N-terminal 120 residues, is highly expressed. Here, we describe a protocol for the recombinant overexpression and purification of untagged HBx_1-120 from bacterial cells. The procedure is compatible with stable isotope labelling in minimal media. Following cell lysis, HBx_1-120 was recovered from inclusion bodies (IBs), solubilized in urea, and purified by ion-exchange (IEX) and size-exclusion chromatography (SEC). The purified protein was extensively characterized, including by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Functionality was confirmed by a pulldown assay with a known interacting partner, Spindlin1. This protocol provides a robust framework to obtain untagged HBx_1-120 for structural and functional in vitro studies.

人类乙型肝炎病毒（HBV）引起乙型肝炎，这是一种急性或慢性肝脏感染。HBV编码四种蛋白，其中X蛋白（HBx）在病毒复制中起关键作用。在慢性HBV感染期间，病毒DNA被整合到宿主基因组中，包含n端120残基的HBx1-120亚型被高度表达。在这里，我们描述了一种从细菌细胞中重组过表达和纯化未标记HBx1-120的方案。该方法与稳定同位素标记在最小介质中兼容。细胞裂解后，从包涵体（IBs）中回收HBx1-120，在尿素中溶解，并通过离子交换（IEX）和尺寸排除色谱（SEC）纯化。纯化后的蛋白被广泛地表征，包括质谱和核磁共振（NMR）光谱。功能通过与已知的相互作用伙伴Spindlin1的下拉试验确认。该方案为获得用于体外结构和功能研究的未标记HBx1-120提供了一个强大的框架。

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引用次数: 0

Catalytically active hCELA3B is a natively-folded monomer and is N-glycosylated 具有催化活性的hCELA3B是一种天然折叠单体，并被n -糖基化。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-02 DOI: 10.1016/j.pep.2025.106840

Prince Kumar , Prabhakar Babele , Rishav Madhukalya , Abhishek Goswami , Khadijah Ameen Khan , Rohit Gupta , Anica Dadwal , Rajesh Kumar , Dilip Kumar , Pramod Kumar Garg , Supratik Das

Chymotrypsin-like elastases CELA3A and CELA3B constitute a sub-family of serine proteases that hydrolyze proteins such as elastin. They are secreted from the pancreas as zymogens, are activated catalytically after cleavage by trypsin, and have digestive function in the intestine. A decrease in these enzymes, due to pancreatitis, can lead to pancreatic exocrine insufficiency (PEI). However, the properties and structures of human CELA3A and CELA3B remain to be determined. To address this, we developed a method to express and purify pro-hCELA3A and pro-hCELA3B from mammalian suspension cultures. We report that the protein can be rapidly purified from the supernatant of expression cultures to apparent homogeneity. The proteins are natively folded and enzymatically active. Only pro-hCELA3B forms homogeneous monomers making it suitable for further structural studies. Pro-hCELA3B is N-glycosylated.

凝乳胰蛋白酶样弹性酶CELA3A和CELA3B是丝氨酸蛋白酶的一个亚家族，可水解弹性蛋白等蛋白质。它们作为酶原从胰腺分泌，经胰蛋白酶裂解后被催化激活，并在肠内具有消化功能。这些酶的减少，由于胰腺炎，可导致胰腺外分泌功能不全（PEI）。然而，人类CELA3A和CELA3B的性质和结构仍有待确定。为了解决这个问题，我们开发了一种从哺乳动物悬浮培养中表达和纯化pro-hCELA3A和pro-hCELA3B的方法。我们报道该蛋白可以从表达培养的上清中快速纯化到明显的均匀性。这些蛋白质是天然折叠的，具有酶活性。只有pro-hCELA3B形成均相单体，适于进一步的结构研究。Pro-hCELA3B被n -糖基化。

引用次数: 0

Recombinant expression of antimicrobial peptide AMP-PD as tandem octamer and its antibacterial activity in vitro 抗菌肽AMP-PD串联八聚体的重组表达及其体外抗菌活性研究。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-10-23 DOI: 10.1016/j.pep.2025.106839

Xingmiao Lu , Yuan Sun , Ziyi Wang , Xiaoke Jin , Jiayu Zhang , Fuping Lu , Wen-Chao Yang , Fufeng Liu

The widespread misuse of antibiotics has led to increased bacterial resistance, posing a severe threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternatives due to their low resistance and broad-spectrum antibacterial activity. However, the low heterologous expression efficiency of AMPs has hindered their large-scale application. Herein, a novel tandemly gene duplication expression system of AMP was developed with recombinant plasmid His₆-AMP-PD₈ that containing eight OM19R repeats and tandemly linked by aspartic acid (D) and proline (P), using AMP-PD (PVDKPPYLPRPRPIRRPGGRD) as a model. Soluble expression of the fusion protein was successfully achieved in E. coli BL21(DE3). The expression efficiency of the fusion protein, including induction temperature, induction time, and IPTG concentration, was optimized to enhance the yield of the recombinant protein. About 32 mg/L the recombinant protein was achieved under the optimal condition, 35 °C, 0.7 mmol/L IPTG and 10 h of induction time. Then, 16 mg/L AMP-PD was obtained after cleavage with 50 % formic acid at 55 °C for 24 h and isolated using 3 kDa ultrafiltration device. Finally, the AMP-PD exhibited considerable antibacterial activity with 12.59 mm of inhibition circle diameter, and the MIC and MBC for E. coli ATCC25922 were 45.75 μg/mL and 62.53 μg/mL, respectively. This study successfully realized the soluble expression system of AMP-PD, laying the foundation for its industrial production and general application for AMP biosynthesis.

抗生素的广泛滥用导致细菌耐药性增强，对公众健康构成严重威胁。抗菌肽（AMPs）由于其低耐药性和广谱抗菌活性而成为有前途的替代品。然而，AMPs的低异种表达效率阻碍了其大规模应用。本文以AMP- pd （PVDKPPYLPRPRPIRRPGGRD）为模型，以含有8个OM19R重复序列、由天冬氨酸(D)和脯氨酸(P)串联连接的重组质粒His6-AMP-PD8为载体，构建了AMP基因串联复制表达体系。在大肠杆菌BL21（DE3）中成功实现了融合蛋白的可溶性表达。优化融合蛋白的表达效率，包括诱导温度、诱导时间和IPTG浓度，以提高重组蛋白的产量。在35℃、0.7 mmol/L IPTG和10 h的诱导条件下，重组蛋白的表达量约为32 mg/L。然后用50%甲酸在55℃下裂解24 h，得到16 mg/L的AMP-PD，用3kda超滤装置分离。结果表明，AMP-PD对大肠杆菌ATCC25922的抑制圈直径为12.59 mm， MIC和MBC分别为45.75 μg/mL和62.53 μg/mL。本研究成功实现了AMP- pd的可溶性表达体系，为AMP生物合成的工业化生产和普遍应用奠定了基础。

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引用次数: 0

The long chain fatty acid-CoA ligase VraA plays a regulatory role in vancomycin resistance in Staphylococcus aureus 长链脂肪酸-辅酶a连接酶VraA在金黄色葡萄球菌万古霉素耐药过程中起调节作用。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-10-21 DOI: 10.1016/j.pep.2025.106838

Dafeng Liu , Na Li

The pathogen Staphylococcus aureus threatens clinical and public health. Vancomycin, a former last-resort treatment, has waning efficacy against resistant strains (VISA) that alter their cell envelope using enzymes like VraA. However, the specific functional mechanism of VraA in VISA is still not understood. Here, we successfully obtained monomeric VraA and found that oleate is the optimal substrate for VraA. VraA was characterized as a peripheral membrane protein with a hydrodynamic radius of 5.7 ± 0.3 nm. Disruption of VraA significantly increased S. aureus susceptibility to vancomycin, as demonstrated by minimum inhibitory concentration (MIC) assays and RT-qPCR. The structural model of VraA predicted using AlphaFold2 was refined via ModRefiner, and active site residues were identified. Our findings underscore the pivotal role of VraA in mediating vancomycin resistance in S. aureus, providing valuable insights for developing targeted therapies against this pathogen.

病原菌金黄色葡萄球菌威胁着临床和公众健康。万古霉素曾是一种最后的治疗手段，但它对利用VraA等酶改变细胞包膜的耐药菌株（VISA）的疗效正在减弱。然而，VraA在VISA中的具体作用机制尚不清楚。在这里，我们成功地获得了单体VraA，并发现油酸酯是VraA的最佳底物。VraA为外周膜蛋白，水动力半径为5.7±0.3 nm。最低抑制浓度（MIC）测定和RT-qPCR结果表明，破坏VraA可显著增加金黄色葡萄球菌对万古霉素的敏感性。通过ModRefiner对AlphaFold2预测的VraA结构模型进行细化，并鉴定出活性位点残基。我们的发现强调了VraA在介导金黄色葡萄球菌万古霉素耐药性中的关键作用，为开发针对该病原体的靶向治疗提供了有价值的见解。

引用次数: 0

Effect of culture media and fermentation process on the refolding and purification of rh-GCSF 培养基和发酵工艺对rh-GCSF再折叠和纯化的影响。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-10-21 DOI: 10.1016/j.pep.2025.106837

Somayeh Abolghasemi , Fatemeh Poureini , Valiollah Babaeipour , Faezeh Farji , Mohammad Reza Mofid

Human granulocyte-colony stimulating factor (h-GCSF) is used to mitigate neutropenia caused by chemotherapy. E. coli is the most common host for h-GCSF production due to its high yield and versatile culture strategies. However, overproduction of h-GCSF in E. coli often results in the formation of inclusion bodies (IBs) in the cytoplasm. The efficiency of refolding and purifying recombinant h-GCSF (rh-GCSF) produced as IBs depends on the expression strategy, induction, and cell growth conditions. This study investigated how different culture media and fermentation processes affect the refolding of IBs and the purification of rh-GCSF. The purified samples were compared to Neupogen® and PDgrastim reference standards using Western blotting, size exclusion chromatography, and reverse-phase high-performance liquid chromatography. The analysis confirmed that all proteins were correctly refolded and exhibited a purity exceeding 90 % after purification. The highest protein recovery percentage and purity of rh-GCSF with the lowest endotoxin content was achieved using M9 media in fed-batch culture.

人粒细胞集落刺激因子（h-GCSF）用于减轻化疗引起的中性粒细胞减少症。大肠杆菌是生产h-GCSF最常见的宿主，因为它的高产和多样化的培养策略。然而，大肠杆菌中h-GCSF的过量产生经常导致细胞质中包涵体（IBs）的形成。重组h-GCSF （rh-GCSF）以IBs的形式产生，其重折叠和纯化的效率取决于表达策略、诱导和细胞生长条件。本研究考察了不同培养基和发酵工艺对IBs再折叠和rh-GCSF纯化的影响。纯化后的样品与Neupogen®和PDgrastim标准品进行Western blotting、排色层析和反相高效液相色谱比较。分析证实所有蛋白都正确折叠，纯化后纯度超过90%。采用M9培养基进行分批补料培养，获得的rh-GCSF蛋白回收率和纯度最高，内毒素含量最低。

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引用次数: 0