Protein expression and purification最新文献_第10页

Column-free purification of functional HIV-1 capsid protein and its application in assembly and inhibitor assays 功能性HIV-1衣壳蛋白的无柱纯化及其在组装和抑制剂检测中的应用

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-28 DOI: 10.1016/j.pep.2025.106766

Da-Wei Zhang , Xiao-Shuang Xu , Yimin Li , Shan Chang

The HIV-1 capsid protein (CA) is essential for viral replication and serves as a validated antiviral drug target. Traditional purification of CA relies on multi-step chromatographic protocols, which are time-consuming and labor-intensive. In this study, we established a rapid, column-free purification strategy using a cleavable self-aggregating tag (cSAT) to produce functional wild-type CA protein from E. coli with >95 % purity within a single day. The workflow is compatible with high-throughput formats and scalable from microplates to fermenters, offering significant advantages over conventional purification methods. The purified CA retained full biological activity, as demonstrated by its ability to assemble into higher-order structures in a salt- and protein concentration–dependent manner in vitro. We further evaluated the effects of two well-characterized capsid modulators: CAI, a peptide inhibitor, and lenacapavir (LEN), a clinically approved capsid-targeting drug. Turbidity-based assembly assays confirmed that CAI inhibited and LEN enhanced CA assembly in a dose-dependent manner. When co-administered, CAI and LEN exhibited mutually antagonistic effects. Preincubation with CAI abolished LEN-mediated enhancement, indicating a potential conformational lock imposed by CAI. These findings demonstrate that the column-free strategy enables efficient production of functionally active CA protein suitable for downstream biochemical and inhibitor screening assays. The approach provides a practical tool for accelerating HIV-1 capsid research and antiviral discovery.

HIV-1衣壳蛋白（CA）对病毒复制至关重要，是一种有效的抗病毒药物靶点。传统的CA纯化依赖于多步骤色谱协议，耗时费力。在这项研究中，我们建立了一种快速的无柱纯化策略，使用可切割自聚集标签（cSAT）在一天内从大肠杆菌中获得95%纯度的功能性野生型CA蛋白。该工作流程与高通量格式兼容，可从微孔板扩展到发酵罐，与传统纯化方法相比具有显着优势。纯化后的CA保留了充分的生物活性，这证明了它在体外以盐和蛋白质浓度依赖的方式组装成高阶结构的能力。我们进一步评估了两种表征良好的衣壳调节剂的作用：肽抑制剂CAI和临床批准的衣壳靶向药物lenacapavir （LEN）。基于浊度的组装试验证实CAI以剂量依赖的方式抑制和LEN增强CA组装。同时给药时，CAI和LEN表现出相互拮抗作用。CAI的预培养消除了len介导的增强，表明CAI施加了潜在的构象锁。这些发现表明，无柱策略可以有效地生产功能活跃的CA蛋白，适用于下游生化和抑制剂筛选试验。该方法为加速HIV-1衣壳研究和抗病毒药物的发现提供了实用的工具。

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引用次数: 0

Co-expression enhances cloning efficiency and protein production in CHO cells 共表达提高CHO细胞的克隆效率和蛋白产量。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-18 DOI: 10.1016/j.pep.2025.106763

Yan Fang, Lani Shi, Yan Wang, Congcong Jin, Zhen Sun, Xi Chen, Kang Zhang, Jie Chen, Jiali Qi

Chinese hamster ovary (CHO) cell lines are widely used in the biopharmaceutical industry, particularly for the production of monoclonal antibodies and recombinant protein drugs. Recombinant cell lines are typically derived from single-cell clones to ensure product consistency and stability. However, the difficult-to-express recombinant proteins may impair single-cell proliferation, thereby significantly reducing cloning efficiency. In this study, co-expression of insulin-like growth factor-1 (IGF-1) was used to improve the single-cell cloning efficiency. Co-expression of IGF-1 significantly improved bulk pool growth and increased cloning efficiency by 53–196 %. Batch cell culture evaluation studies further demonstrated that IGF-1 co-expression enhanced monoclonal cell density by 29–64 % and recombinant protein yield by 47–89 %. Additionally, the growth profile, titer, and critical quality attributes of selected cell clones remained stable over 60 generations. The results of this study demonstrate that IGF-1 co-expression represents an effective single-cell cloning strategy by significantly enhancing cloning efficiency and enabling stable production of difficult-to-express proteins.

中国仓鼠卵巢（CHO）细胞系广泛应用于生物制药行业，特别是用于生产单克隆抗体和重组蛋白药物。重组细胞系通常来源于单细胞克隆，以确保产品的一致性和稳定性。然而，难以表达的重组蛋白可能会损害单细胞的增殖，从而显著降低克隆效率。本研究通过胰岛素样生长因子-1 （IGF-1）的共表达来提高单细胞克隆效率。共表达IGF-1显著提高了体池生长，克隆效率提高了53-196%。批量细胞培养评价研究进一步表明，IGF-1共表达可使单克隆细胞密度提高29-64%，重组蛋白产量提高47-89%。此外，所选细胞克隆的生长特征、滴度和关键质量属性在60代以上保持稳定。本研究结果表明，IGF-1共表达是一种有效的单细胞克隆策略，可以显著提高克隆效率并稳定地产生难以表达的蛋白。

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引用次数: 0

From Silkroad to Bioroad–Silkworm Biotechnology– 从丝绸之路到生物之路——蚕生物技术。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-16 DOI: 10.1016/j.pep.2025.106762

Enoch Y. Park

Silkworms have shared history with humans for 5000 years and have significantly contributed to human welfare. Silkworms' ability to produce silk has attracted significant attention as an innovative protein expression system in 21st-century biotechnology, leading to the development of a protein production facility known as the silkworm biofactory. Unlike Escherichia coli expression systems, silkworms can produce recombinant proteins with the added benefits of post-translational modification, easy scalability, and low production costs, as they do not require bioreactors or specialized facilities and expensive media. Numerous recombinant proteins, including secretory and membrane proteins and virus-like particles (VLPs), have been successfully expressed in silkworms and purified, demonstrating their biological functions. Additionally, purification methods have been developed to manufacture recombinant proteins in silkworms. This review provides a comprehensive overview of the silkworm expression system, tracing its development from the past to the present. It highlights advancements in bioengineering related to the production of enveloped and non-enveloped VLPs. Furthermore, the review discusses the technology for displaying antigens on the surface of VLPs, aiming to improve vaccine efficacy through surface conjugations.

蚕与人类共享了5000年的历史，为人类的福祉做出了重大贡献。蚕的产丝能力作为21世纪生物技术中一种创新的蛋白质表达系统引起了极大的关注，导致了一种被称为蚕生物工厂的蛋白质生产设施的发展。与大肠杆菌表达系统不同，家蚕可以生产重组蛋白，并且具有翻译后修饰，易于扩展和低生产成本的额外优点，因为它们不需要生物反应器或专门的设施和昂贵的培养基。许多重组蛋白，包括分泌蛋白和膜蛋白以及病毒样颗粒（vlp），已经在家蚕中成功表达和纯化，并证明了它们的生物学功能。此外，已经开发了纯化方法来制造家蚕重组蛋白。本文对家蚕表达系统进行了全面的综述，追溯了其从过去到现在的发展历程。它强调了与包膜和非包膜VLPs生产相关的生物工程进展。此外，本文还讨论了在VLPs表面展示抗原的技术，旨在通过表面偶联来提高疫苗的效力。

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引用次数: 0

Molecular chaperones: A revolutionary approach for increased solubility of recombinant mAbs from bacterial and yeast systems 分子伴侣：从细菌和酵母系统中增加溶解度的重组单克隆抗体的革命性方法

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-13 DOI: 10.1016/j.pep.2025.106764

Niharika Ashish Kulkarni, Prabir Kumar Das, Arjun P, Venkata Dasu Veeranki

Recombinant protein expression has revolutionized biotechnology, enabling the production of therapeutic proteins, enzymes, and antibodies with high specificity and functionality. Escherichia coli can be a prominent host for recombinant protein production due to its rapid growth, well-characterized genetics, and cost-effectiveness. However, the formation of insoluble protein aggregates, misfolding, and stability issues are significant challenges. This review explores the integration of molecular chaperones such as GroEL/GroES, DnaK/DnaJ/GrpE, and Skp in recombinant systems to enhance folding, solubility, and activity of recombinant monoclonal antibodies (mAbs). It also examines advances in co-expression strategies, secretion pathways, and the role of engineered host strains in overcoming these bottlenecks. Further, the review highlights comparative approaches in other expression systems in yeast, including Saccharomyces cerevisiae and Pichia pastoris, focusing on the molecular chaperone-assisted folding of full-length and fragmented mAbs using Hsp70-90 (analogs of DnaJ and K, respectively), tailless complex polypeptide 1 ring complex (TRiC), and ribosome-associated complex (RAC)/nascent polypeptide-associated complex (NAC) system. Innovations such as the application of polymer nanoparticles as artificial chaperones and mRNA engineering for co-translational folding underscore the potential for optimizing recombinant protein production. Collectively, these findings offer a thorough grasp of the role of chaperones and engineering strategies in improving mAbs quality, with implications for biopharmaceutical manufacturing and industrial applications.

重组蛋白表达彻底改变了生物技术，使生产具有高特异性和功能性的治疗蛋白、酶和抗体成为可能。大肠杆菌由于其快速生长、良好的遗传特征和成本效益，可以成为重组蛋白生产的重要宿主。然而，不溶性蛋白质聚集体的形成、错误折叠和稳定性问题是重大挑战。本文综述了分子伴侣如GroEL/GroES， DnaK/DnaJ/GrpE和Skp在重组系统中的整合，以增强重组单克隆抗体（mab）的折叠性，溶解度和活性。它还研究了共表达策略、分泌途径和工程宿主菌株在克服这些瓶颈中的作用方面的进展。此外，本文还重点介绍了在酵母中其他表达系统（包括酿酒酵母和毕氏酵母）中的比较方法，重点研究了使用Hsp70-90（分别是DnaJ和K的类似物）、无尾复合体多肽1环复合体（TRiC）和核糖体相关复合体(RAC)/新生儿多肽相关复合体（NAC）系统进行全长和片段化单克隆抗体的分子伴侣辅助折叠。聚合物纳米颗粒作为人工伴侣的应用和mRNA工程用于共翻译折叠等创新强调了优化重组蛋白生产的潜力。总的来说，这些发现提供了对伴侣蛋白和工程策略在提高单克隆抗体质量中的作用的全面把握，对生物制药制造和工业应用具有重要意义。

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引用次数: 0

Efficient synchronization of high expression and site-specific biotinylation of the PTBP1 RRM3/4 domain PTBP1 RRM3/4结构域的高表达和位点特异性生物素化的高效同步

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-05 DOI: 10.1016/j.pep.2025.106755

Zhangheng Ye , Hong Gu , Yuanhang Wang, Yingyi Liu, Yilan Huang, Meiyi Mao, Lan Sun, Mingxing Huang

Polypyrimidine tract-binding protein 1 (PTBP1), a key regulator of mRNA alternative splicing, has emerged as a promising therapeutic target for neurodegenerative diseases and cancer treatment. Given the critical role of its RRM4 domain's lysine residues in binding to polypyrimidine single-stranded RNA, nonspecific biotinylation could potentially alter PTBP1's activity. Here, we describe a method for the concurrent high-level expression and site-specific biotinylation of the PTBP1 RRM3/4 domain using the pQE80L vector and AVB101 host strain. By optimizing codon usage and GC content, we achieved a maximum expression level of approximately 85 μg/mL in bacterial culture, with over 99 % purity and a 72.4 % biotinylation rate. The recombinant 6His-Avi^Biotin-PTBP1 demonstrated the ability to emit chemiluminescence when coupled with HRP-streptavidin, to be immunoprecipitated by streptavidin magnetic beads, and to interact specifically with (CU)₈ RNA, exhibiting a dissociation constant (K_D) of 38 nM as determined by Bio-Layer Interferometry (BLI). Collectively, these results indicate that the recombinant 6His-Avi^Biotin-PTBP1 is suitable for inhibitor screening and kinetic parameter analysis.

多嘧啶束结合蛋白1 （PTBP1）是mRNA选择性剪接的关键调节因子，已成为神经退行性疾病和癌症治疗的一个有希望的治疗靶点。鉴于其RRM4结构域赖氨酸残基与多嘧啶单链RNA结合的关键作用，非特异性生物素化可能潜在地改变PTBP1的活性。在这里，我们描述了一种利用pQE80L载体和AVB101宿主菌株同时高水平表达PTBP1 RRM3/4结构域和位点特异性生物素化的方法。通过优化密码子的使用和GC含量，我们在细菌培养中获得了约85 μg/mL的最大表达量，纯度超过99%，生物素化率为72.4%。重组6His-AviBiotin-PTBP1与HRP-streptavidin偶联时具有化学发光的能力，可被streptavidin磁珠免疫沉淀，并与（CU）8 RNA特异性相互作用，通过生物层干涉法（BLI）测定其解离常数（KD）为38 nM。综上所述，这些结果表明重组6His-AviBiotin-PTBP1适合用于抑制剂筛选和动力学参数分析。

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引用次数: 0

Efficient production of functional proaerolysin in E. coli 大肠杆菌中功能性原溶素的高效生产。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-04 DOI: 10.1016/j.pep.2025.106754

Quynh Thi-Huong Pham , Ayako Tagawa , Narumi Iwata , Masaru Nakao , Yusuke Miyanari

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, forming transmembrane pores that induce cell death. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA, a gene involved in the biosynthesis of GPI anchors. Beyond diagnostics, proaerolysin has recently been applied in basic research, including its use as a counter-selection agent in genetic manipulations and as an engineered nanopore for single-molecule detection. Although the bacterial expression and purification of proaerolysin have been previously reported, the yields were low due to its low solubility. Here, we demonstrate that using the SHuffle E. coli strain, which facilitates the disulfide bond formation in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 mL bacterial culture, with a purity exceeding 99 %. The functionality of recombinant proaerolysin was confirmed through testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method provides a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.

原气溶素是由嗜水气单胞菌产生的一种细菌毒素，它特异性地与质膜上gpi锚定的蛋白质结合，形成跨膜孔，诱导细胞死亡。利用这种独特的特性，proaerysis被广泛用于阵发性夜间血红蛋白尿（PNH）的诊断测试，PNH是由PIGA（一种参与GPI锚定生物合成的基因）的体细胞突变引起的疾病。除了诊断之外，proaerlysin最近还被应用于基础研究，包括在基因操作中用作反选择剂，以及作为单分子检测的工程纳米孔。虽然以前已经报道过细菌表达和纯化原气溶素，但由于其溶解度低，产量很低。在这里，我们证明了使用SHuffle大肠杆菌菌株，促进细胞质中二硫键的形成，显著提高原气溶素的溶解度和正确折叠。我们获得了高产量的proprolysin，大约3毫克从50毫升的细菌培养，纯度超过99%。通过小鼠胚胎干细胞（mESCs）的测试证实了重组原气溶素的功能，表明这种高产的生产方法为广泛的生物技术应用提供了可靠和经济的功能性原气溶素来源。

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引用次数: 0

Strategies for generating soluble and monomeric samples of Ycf1p NBD2 生成Ycf1p NBD2可溶性和单体样品的策略

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-03 DOI: 10.1016/j.pep.2025.106752

Sarah E.S. Quail , Jeffrey Youn , Voula Kanelis

The yeast cadmium factor 1 protein (Ycf1p) is an ATP-binding cassette (ABC) transporter that is located in the vacuolar membrane and is responsible for transporting glutathione-conjugated metals from the cytoplasm into the vacuole. Ycf1p contains the ABC core structure of two transmembrane domains (TMD1, TMD2) and two nucleotide-binding domains (NBD1, NBD2). As a member of the C-subfamily of ABC proteins (ABCC), Ycf1p also contains an N-terminal extension comprised of an additional TMD (TMD0) and L0 linker. Although high-resolution structures of many ABC transporters have been determined, the NBDs can be at low resolution in cryo-EM maps. Thus, studies of the isolated cytosolic NBDs are crucial for obtaining molecular-level details of the dynamics of these catalytic entities, for example. In this study, we present a scheme for obtaining samples of NBD2 from the yeast cadmium factor 1 protein (Ycf1p) in a soluble, monomeric, and functional form. While production of NBD1 from Ycf1p and other ABC proteins has been accomplished, generating samples of NBD2 from different ABC proteins has been elusive for the most part, particularly for ABCC proteins. We show that NBD2 preparation necessitates minimizing dimerization and aggregation of the protein at multiple steps during the purification, which is accomplished by employing a solubility tag, eliminating nucleotides from the buffers, and limiting the duration of spin concentrating steps. This work lays the foundation for detailed studies of Ycf1p NBD2 and provides an outline for optimizing the generation of samples of NBD2 from other ABC proteins.

酵母镉因子1蛋白（Ycf1p）是一种atp结合盒（ABC）转运蛋白，位于液泡膜上，负责将谷胱甘肽结合的金属从细胞质转运到液泡中。Ycf1p含有两个跨膜结构域（TMD1、TMD2）和两个核苷酸结合结构域（NBD1、NBD2）的ABC核心结构。作为ABC蛋白c亚家族（ABCC）的成员，Ycf1p还含有一个n端延伸，由一个额外的TMD （TMD0）和L0连接子组成。虽然许多ABC转运体的高分辨率结构已经确定，但在低温电镜图中，nbd可能是低分辨率的。因此，对分离胞质nbd的研究对于获得这些催化实体的分子水平动力学细节至关重要。在这项研究中，我们提出了一种从酵母镉因子1蛋白（Ycf1p）中以可溶性、单体和功能形式获得NBD2样品的方案。虽然从Ycf1p和其他ABC蛋白中产生NBD1已经完成，但从不同的ABC蛋白中产生NBD2的样品在很大程度上是难以捉摸的，特别是对于ABCC蛋白。我们发现NBD2的制备需要在纯化过程中的多个步骤中尽量减少蛋白质的二聚化和聚集，这是通过使用溶解度标签，从缓冲液中去除核苷酸，并限制自旋浓缩步骤的持续时间来完成的。这项工作为Ycf1p NBD2的详细研究奠定了基础，并为优化从其他ABC蛋白中生成NBD2样品提供了大纲。

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引用次数: 0

Expression, purification and functional study of mycobacteriophage D29 histidine-asparagine-histidine endonuclease 分支噬菌体D29组氨酸-天冬酰胺-组氨酸内切酶的表达、纯化及功能研究。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-06-02 DOI: 10.1016/j.pep.2025.106753

Bo Fu, Lvming Wu, Xin Wang, Hongbin Sun

Mycobacteriophage histidine-asparagine-histidine endonuclease (mpHNHE) is a protein encoded by mycobacteriophage D29, featuring a conserved HNH motif and belonging to the HNH nuclease superfamily. To explore its physiological functions, the recombinant plasmid pET-28a (+)-mpHNHE was constructed and expressed in E. coli BL21 (DE3). The inclusion body form of the expression product was purified using urea denaturation combined with nickel affinity chromatography and gel filtration chromatography. Structural characterization revealed that mpHNHE exists as a monomer in solution, predominantly composed of β-sheets, and exhibits good structural stability. Enzymatic property studies indicated that mpHNHE has high nuclease activity, significant substrate size selectivity, and metal ion dependence. These findings not only provide new insights into the structure-function relationship of HNH-type nucleases but also provide a molecular basis for the development of new nuclease tools and lay the foundation for understanding the mechanism of mpHNHE in D29.

分枝噬菌体组氨酸-天冬酰胺-组氨酸内切酶（mpHNHE）是一种由分枝噬菌体D29编码的蛋白，具有保守的HNH基序，属于HNH核酸酶超家族。为探究其生理功能，构建了重组质粒pET-28a(+)-mpHNHE，并在大肠杆菌BL21（DE3）中表达。采用尿素变性结合镍亲和层析和凝胶过滤层析对表达产物的包涵体形态进行了纯化。结构表征表明，mpHNHE在溶液中以单体形式存在，主要由β-片组成，具有良好的结构稳定性。酶学性质研究表明，mpHNHE具有高核酸酶活性，显著的底物大小选择性和金属离子依赖性。这些发现不仅为hnh型核酸酶的结构-功能关系提供了新的见解，而且为开发新的核酸酶工具提供了分子基础，并为理解mpHNHE在D29中的作用机制奠定了基础。

引用次数: 0

Bioinformatics analysis and molecular cloning of squalene synthase from Simaroubaceae 角鲨烯合成酶的生物信息学分析及分子克隆。

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-05-30 DOI: 10.1016/j.pep.2025.106751

Yiqing Zhu , Shiyu Wu , Yi Zhang , Shiyang Zhang , Qianyu Zhou , Weidong Zhang , Jinxin Wang , Zhimin Hu

Quassinoids are a class of highly oxygenated triterpenoids with C-18, C-19, C-20, C-22, or C-25 skeletons. Squalene synthase (SQS), a key enzyme in the quassinoids biosynthetic pathway, serves as the first step in controlling carbon flux flow into downstream quassinoids. In this study, we screened and analyzed the SQSs from the transcriptome data of the Simaroubaceae family, including AaSQS, BjSQS, AeSQS, QaSQS, ElSQS, and SaSQS. Bioinformatics analysis showed that these SQSs contain C-terminal transmembrane regions and exhibit over 80 % sequence identity with that of Glycyrrhiza glabra (GgSQS1). Phylogenetic analysis revealed that the SQSs from the Simaroubaceae family are more closely related to Malus domestica and Crataegus pinnatifida within the Rosales clade, suggesting an evolutionary trajectory that corroborates the taxonomic classification of Simaroubaceae within the Rosanae superorder. To characterize the function of the SQS from the Simaroubaceae family, the full-length AaSQS was cloned. A soluble AaSQS was obtained by expressing the truncated version lacking 24 amino acids at the C-terminal region in Escherichia coli. Functional analysis showed that AaSQS could catalyze the production of squalene from farnesyl pyrophosphate (FPP). Structural prediction and molecular docking indicated that residues S50, F51, L205, N209, R212, C286, P289, and M313 may be key catalytic residues in AaSQS. Further mutation analysis revealed that all mutants except C286A showed reduced squalene accumulation compared to the wild type (WT). These results provide guidance for quassinoid biosynthesis through metabolic engineering and synthetic biology strategies, particularly by engineering key amino acid residues to enhance enzymatic activity. This study advances the understanding of SQSs in the Simaroubaceae family and provides an important foundation for the study of quassinoid biosynthesis.

拟assinoids是一类具有C-18， C-19, C-20， C-22或C-25骨架的高氧三萜化合物。角鲨烯合成酶（Squalene synthase， SQS）是类星体生物合成途径中的关键酶，是控制碳通量流向下游类星体的第一步。在本研究中，我们从simmaroubacae科的转录组数据中筛选并分析了sqs，包括AaSQS、BjSQS、AeSQS、QaSQS、ElSQS和SaSQS。生物信息学分析表明，这些序列包含c端跨膜区，与Glycyrrhiza glabra （GgSQS1）序列同源性超过80%。系统发育分析表明，Simaroubaceae科的SQSs与Rosales分支中的Malus domestica和Crataegus pinnatifida亲缘关系更近，这一进化轨迹证实了Simaroubaceae在Rosanae超目中的分类学分类。为了进一步确定该基因的功能，对该基因进行了克隆。通过在大肠杆菌中表达c端缺失24个氨基酸的片段，获得了可溶性的AaSQS。功能分析表明，AaSQS可催化法尼酯焦磷酸（FPP）合成角鲨烯。结构预测和分子对接表明，S50、F51、L205、N209、R212、C286、P289和M313可能是AaSQS的关键催化残基。进一步的突变分析表明，与野生型（WT）相比，除C286A外，所有突变体的角鲨烯积累量都有所减少。这些结果为通过代谢工程和合成生物学策略，特别是通过工程设计关键氨基酸残基来增强酶活性提供了指导。该研究促进了人们对拟南芥科植物SQSs的认识，为拟南芥的生物合成研究提供了重要的基础。

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引用次数: 0

Expression and purification of functional recombinant Cullin1-Rbx1/2 in E. coli 功能性重组Cullin1-Rbx1/2在大肠杆菌中的表达与纯化

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-05-28 DOI: 10.1016/j.pep.2025.106750

Fangyu Zhao , Chuntong Li , Xu Li , Luyu Shi , Yingyue Zhang , Han Wang , Shuzhe Sun , Lu-Jun Liang

Protein ubiquitination is a crucial post-translational modification in eukaryotes that is mediated by E1-E2-E3 enzymatic cascades and regulates a wide range of cellular processes. Cullin-RING E3 ligases (CRLs) represent the largest family of RING-type E3 ligases and play pivotal roles in these processes. Generating full-length, active Cullin1-Rbx1 (CRL1) is essential for biochemical and biophysical investigations. In this study, we developed an efficient strategy to produce functional CRL1 complexes from E. coli, by fusing an MsyB protein to N-terminus of Cullin1 to improve its solubility. The recombinant CRL1 demonstrated full functionality, successfully assembling with substrate receptor Skp1-Skp2-Cks1 and mediating the polyubiquitination of Ub-p27-degron substrate. Using recombinant CRL1, we found that phosphorylation at Ser65 inhibited the CRL1-UBE2R1 mediated ubiquitin chain elongation on Ub-p27-degron. Furthermore, using the same strategy, we successfully generated Cullin1-Rbx1 mutants and Cullin1-Rbx2 complexes, thereby expanding the applicability of our method. Collectively, this work establishes a rapid and cost-effective platform for the production of CRL1 complexes, facilitating structural and functional studies.

蛋白泛素化是真核生物中一个重要的翻译后修饰，由E1-E2-E3酶级联介导，并调节广泛的细胞过程。Cullin-RING E3连接酶（CRLs）是最大的ring - E3连接酶家族，在这些过程中起着关键作用。产生全长、活性的Cullin1-Rbx1 （CRL1）对于生物化学和生物物理研究至关重要。在这项研究中，我们开发了一种从大肠杆菌中产生功能性CRL1复合物的有效策略，通过将MsyB蛋白融合到Cullin1的n端来提高其溶解度。重组CRL1显示出完整的功能，成功地与底物受体Skp1-Skp2-Cks1组装，并介导Ub-p27-degron底物的多泛素化。利用重组CRL1，我们发现Ser65位点的磷酸化抑制了CRL1- ube2r1介导的ub -p27 degron上泛素链的延伸。此外，使用相同的策略，我们成功地生成了Cullin1-Rbx1突变体和Cullin1-Rbx2复合物，从而扩大了我们方法的适用性。总的来说，这项工作为CRL1复合物的生产建立了一个快速和经济的平台，促进了结构和功能的研究。

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引用次数: 0