Protein expression and purification最新文献_第8页

Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P 未折叠蛋白反应激活因子基因HAC1P的共表达促进毕赤酵母茎菠萝蛋白酶的产生

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-20 DOI: 10.1016/j.pep.2025.106804

Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan

Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant Ananas comosus. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) Hac1 on folding and secretion of recombinant stem bromelain in Pichia pastoris. Stem bromelain gene (BL) from Ananas comosus was codon optimized and expressed in the Pichia pastoris X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, Hac1p was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when HAC1 was co-expressed with stem bromelain (BL). Fed batch studies were performed for both pGAPαBL1 and pGAPBLHAC1 clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones like Kar2p, Sec 63, Pdi, Cne1. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies Pichia pastoris as a robust platform to produce stem bromelain for various industrial applications.

高滴度表达外源蛋白和代谢物会对重组宿主产生多种应激反应。菠萝蛋白酶是一种半胱氨酸蛋白酶，存在于菠萝植物Ananas comosus的茎和果实中。该酶具有广泛的工业应用范围，从食品，保健品，化妆品和制药。本研究旨在研究未折叠蛋白反应调节因子（UPR） Hac1对毕氏酵母中重组茎菠萝蛋白酶折叠和分泌的影响。利用组成型启动子和诱导型启动子在毕赤酵母X-33宿主中进行了菠萝蛋白酶基因（BL）的密码子优化和表达。为了折叠UPR调控因子内质网（ER）中错误折叠的蛋白聚集体，Hac1p在GAP启动子的组成表达下与菠萝蛋白酶共表达。摇瓶研究结果表明，当HAC1与茎菠萝蛋白酶（BL）共表达时，蛋白酶活性增加了2倍，为4 U/mL。在3.7 L KLF生物反应器中，在甘油限制条件下，对pgapap α bl1和pGAPBLHAC1克隆进行了批量投料研究，最高活性分别为8 U/mL和54 U/mL。折叠和分泌通路主要基因的基因表达研究表明，UPR的激活导致Kar2p、Sec 63、Pdi、Cne1等主要伴侣蛋白上调。茎菠萝蛋白酶活性为54 U/mL，是迄今为止文献报道的最高活性。目前的工作表明毕赤酵母作为一个强大的平台生产茎菠萝蛋白酶用于各种工业应用。

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引用次数: 0

Intracytoplasmic sperm injection-mediated strategy for the production of transgenic poultry as a bioreactor 胞浆内精子注射介导的转基因家禽生物反应器生产策略

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-18 DOI: 10.1016/j.pep.2025.106802

Shusei Mizushima

A number of methods are used to produce recombinant proteins, and animal bioreactors have emerged as transgenic systems. Animal bioreactors have the potential to reduce production costs and improve efficiency, thereby providing recombinant proteins that are important for therapeutic applications. Various species, such as goats, cattle, and rabbits, have been genetically modified to serve as bioreactors. Since poultry hens produce more than 300 eggs per year, their eggs may also be used as an efficient platform for the production of protein pharmaceuticals. In addition to traditional viral infection of the blastoderm, the recent development of genome-editing technologies for cultured primordial germ cells has enabled the establishment of transgenic chicken lines via germline chimera production systems. We established a new genome-editing technology combined with the intracytoplasmic sperm injection (ICSI) method that has the potential to produce transgenic quail very efficiently in one generation. The avian ICSI technique and recent advances in genome editing are discussed herein.

许多方法被用来生产重组蛋白，动物生物反应器作为转基因系统已经出现。动物生物反应器具有降低生产成本和提高效率的潜力，从而提供对治疗应用很重要的重组蛋白。各种各样的物种，如山羊、牛和兔子，已经被改造成生物反应器。由于家禽母鸡每年生产300多个鸡蛋，它们的鸡蛋也可以用作生产蛋白质药物的有效平台。除了传统的胚皮病毒感染外，最近发展的用于培养原始生殖细胞的基因组编辑技术使得通过种系嵌合体生产系统建立转基因鸡系成为可能。我们建立了一种新的基因组编辑技术，结合胞浆内单精子注射（ICSI）方法，有可能在一代内非常有效地生产转基因鹌鹑。本文讨论了禽类ICSI技术和基因组编辑的最新进展。

引用次数: 0

Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation 人Gb3/CD77合成酶在昆虫和人细胞中的表达：活性和糖基化的比较

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-18 DOI: 10.1016/j.pep.2025.106803

Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski

Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (α1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: Trichoplusia ni insect cells (High Five) and human embryonic kidney cells (Expi293F™). The High Five cells generate short oligomannose structures, while the Expi293F™ cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F™-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F™ cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F™-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts.

蛋白质的糖基化可以影响它们的折叠、稳定性、运输和酶活性。人Gb3/CD77合成酶（α1,4-半乳糖转移酶，A4galt）具有两个被占用的n -糖基化位点。先前，我们证明了重组酶的活性依赖于它的n -糖基化。在这项研究中，我们在已知具有不同糖基化模式的两种表达宿主：毛癣虫昆虫细胞（High Five）和人胚胎肾细胞（Expi293F™）中制备了人Gb3/CD77合成酶的可溶性重组催化结构域。High Five细胞产生短的寡甘露糖结构，而Expi293F™细胞合成复杂型聚糖。我们评估了High Five衍生酶和Expi293F™衍生酶的活性，表征了它们的n -聚糖结构，并表明High Five细胞提供了更高的酶量和活性。此外，我们使用Expi293F™细胞评估6xhis标签的N端和c端位置，发现只有N端标记的Expi293F™衍生酶显示出活性。相比之下，High Five细胞中产生的酶尽管携带c端标签，但仍具有活性。这些发现强调了糖基化模式和标签位置在不同宿主中产生的人重组糖基转移酶活性中的作用。

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引用次数: 0

Toward reproducible PETase research: A standardized workflow for reliable enzyme production and comparison 迈向可重复的PETase研究：可靠的酶生产和比较的标准化工作流程。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-15 DOI: 10.1016/j.pep.2025.106801

Katerina Jiraskova, Jakub Ptacek, Kristyna Vydra Bousova, Jiri Vondrasek

The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered IsPETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management.

PET酶降解聚对苯二甲酸乙二醇酯（PET）作为一种潜在的塑料废物管理解决方案受到了广泛关注。然而，研究中缺乏标准化的PETase生成方案，这对一致的酶表征和活性比较提出了挑战。不同的生产方法，包括表达系统和纯化技术，可能导致PETase活性的差异。在这里，我们提出了生产野生型和工程型IsPETase变体的统一协议的发展。该协议包括标准化表达，纯化和质量控制步骤，以确保再现性和可靠性。通过更准确地比较PETase变体和解决PETase生产中的不一致性，这种方法促进了合作努力，以推进塑料降解技术，并为加速酶促PET降解及其在塑料废物管理中的应用研究奠定了基础。

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引用次数: 0

Recombinant expression and purification of Plasmodium heme detoxification protein in E. coli: Challenges and discoveries 疟原虫血红素解毒蛋白在大肠杆菌中的重组表达与纯化：挑战与发现。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-05 DOI: 10.1016/j.pep.2025.106789

Rahul Singh , Ravindra D. Makde

Heme, a toxic by-product of Plasmodium's proteolytic digestion of host hemoglobin, is detoxified by the malaria parasite through its conversion into hemozoin (Hz)—the malaria pigment. This detoxification pathway is a key target for many antimalarial drugs, which aim to induce heme-mediated toxicity to the parasite. The Heme Detoxification Protein (HDP) plays a central role in heme-to-Hz transformation; however, its precise mechanism remains unclear, largely due to the absence of successful recombinant expression in a native, soluble form.

In this study, we aimed to express HDP recombinantly in its native soluble state using an E. coli-based system. A range of strategies were employed, including expression of orthologs, consensus sequence design, fusion to solubility-enhancing partners, co-expression with molecular chaperones, and extensive construct optimization through N-terminal truncations. Despite extensive efforts, most recombinant HDP constructs were either insoluble or formed soluble aggregates. Notably, only a single construct—with a 44-residue N-terminal truncation and a C-terminal 6-His tag (HDPpf-C10)—was successfully expressed in a soluble form.

Surprisingly, HDPpf-C10, although retaining domains implicated in heme binding and transformation, exhibited no detectable heme-to-Hz transformation activity. This finding highlights the essential role of the flexible-unstructured N-terminal region in mediating both heme binding and its subsequent conversion to Hz, providing new insights into HDP function and guiding future structural and mechanistic studies.

血红素是疟原虫对宿主血红蛋白进行蛋白水解消化的一种有毒副产物，疟原虫通过将血红素转化为疟原虫色素（Hz）来解毒。这种解毒途径是许多抗疟疾药物的关键靶点，其目的是诱导血红素介导的寄生虫毒性。血红素解毒蛋白（HDP）在血红素-赫兹转化中起核心作用；然而，其确切的机制尚不清楚，这主要是由于缺乏成功的天然可溶形式的重组表达。在这项研究中，我们的目的是在大肠杆菌为基础的系统中重组表达其天然可溶性的HDP。采用了一系列策略，包括表达同源物、共识序列设计、融合到提高溶解度的伙伴、与分子伴侣共表达以及通过n端截断进行广泛的构建优化。尽管经过了广泛的努力，大多数重组HDP构建物要么不溶，要么形成可溶性聚集体。值得注意的是，只有一个单一的结构-具有44个残基的n端截断和c端6×His标签(HDPpf-C10)-成功地以可溶性形式表达。令人惊讶的是，尽管HDPpf-C10保留了与血红素结合和转化有关的结构域，但却没有显示出可检测到的血红素到赫兹的转化活性。这一发现强调了灵活的非结构化n端区域在介导血红素结合及其随后向Hz转化中的重要作用，为HDP功能提供了新的见解，并指导了未来的结构和机制研究。

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引用次数: 0

Ligand multimerization effects on binding efficiency of Protein L affinity chromatography resins 配体多聚对蛋白L亲和层析树脂结合效率的影响

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-08-05 DOI: 10.1016/j.pep.2025.106790

Jafar Nikzad , Kimia Kalantari Khandani , Yeganeh Talebkhan , Fatemeh Zandi , Samira Komijani , Ahmad Adeli

Nearly four decades have passed since the discovery of protein-L. Its exceptional ability in binding to the kappa light chain of immunoglobulins makes it a suitable candidate for the purification of certain biotherapeutics, particularly antibody fragments. Efforts have focused on improving its recovery and dynamic binding capacity. Among various strategies, ligand multimerization has shown significant potential in developing more efficient and cost-effective resins. This study employed a multimerization approach to create new recombinant protein-L-based ligands and compare their performance to the commercially available alternatives. Dynamic binding capacity studies revealed that the engineered ProL6 and ProL8 resins exhibited higher binding capacities than the ProL4 and the commercial Capto-L resin. Furthermore, the recovery rates of Fab antibody fragments from bacterial lysates using ProL6, ProL8, and the commercial resin were 92.8, 94.4, and 94.6 %, respectively, comparable to those of the ProL4 resin. SDS-PAGE analysis confirmed the purity of the proteins eluted from all tested resins, aligned with the results of ProL4 resin. Additionally, it evaluated the 100 % specificity of ProL6, ProL8, and the commercial resins.

蛋白l的发现已经过去了近40年。它与免疫球蛋白的kappa轻链结合的特殊能力使其成为纯化某些生物治疗药物，特别是抗体片段的合适候选者。努力的重点是提高其恢复能力和动态绑定能力。在各种策略中，配体多聚化在开发更高效和更具成本效益的树脂方面显示出巨大的潜力。本研究采用多聚方法创建新的重组蛋白- l基配体，并将其性能与市售替代品进行比较。动态结束力研究表明，设计的ProL6和ProL8树脂比ProL4和Capto-L树脂具有更高的结束力。此外，使用ProL6、ProL8和商用树脂从细菌裂解物中提取Fab抗体片段的回收率分别为92.8%、94.4%和94.6%，与使用ProL4树脂的回收率相当。SDS-PAGE分析证实了从所有测试树脂中洗脱的蛋白质的纯度，与ProL4树脂的结果一致。此外，它还评估了ProL6、ProL8和商用树脂的100%特异性。

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引用次数: 0

Avian primordial germ cell migration: History, mechanisms, applications, and unanswered questions 鸟类原始生殖细胞迁移：历史、机制、应用和未解之谜。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-07-31 DOI: 10.1016/j.pep.2025.106788

Manami Morimoto , Daisuke Saito

Since Swift's discovery of primordial germ cells (PGCs) within the vasculature of chicken embryos in 1914, significant progress has been made in uncovering the origins, migratory pathways, and molecular characteristics of avian PGCs. Recent advances in this field have been accelerated by two synergistic factors: the establishment of robust culture systems that enable efficient genetic manipulation of chicken PGCs, and the inherent suitability of avian embryos for cell transplantation and live imaging. Together, these features have positioned avian PGCs as a powerful system for investigating not only cell migration but also long-standing mysteries in germ cell biology. Furthermore, this system offers a unique platform for dissecting the cellular and molecular mechanisms underlying diseases such as cancer metastasis and germ cell tumors. In this review, we revisit key historical milestones in avian PGC research, explore current knowledge of their migratory regulation, and highlight future directions with potential impact across cell biology, developmental biology, and disease modeling.

自1914年Swift在鸡胚脉管系统中发现原始生殖细胞（PGCs）以来，在揭示鸟类原始生殖细胞的起源、迁移途径和分子特征方面取得了重大进展。两个协同因素加速了这一领域的最新进展：建立强大的培养系统，使鸡PGCs能够有效地进行遗传操作，以及禽胚胎对细胞移植和活体成像的固有适应性。总之，这些特征使禽类PGCs成为一个强大的系统，不仅可以用于研究细胞迁移，还可以用于研究生殖细胞生物学中长期存在的谜团。此外，该系统为剖析癌症转移和生殖细胞肿瘤等疾病的细胞和分子机制提供了一个独特的平台。在这篇综述中，我们回顾了鸟类PGC研究的关键历史里程碑，探索了它们迁移调控的当前知识，并强调了未来的方向，在细胞生物学、发育生物学和疾病建模方面具有潜在的影响。

引用次数: 0

Scalable production of functional recombinant human plasma gelsolin in Escherichia coli for therapeutic and diagnostic applications 在大肠杆菌中大规模生产用于治疗和诊断的功能性重组人血浆凝胶。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-07-30 DOI: 10.1016/j.pep.2025.106786

Young Su Kim , Hye-Jeong Lee , Mi-Reu Kim , Hwabong Jeong , Young Pil Kim , Jung-Ho Park , Jungoh Ahn

Human plasma gelsolin (pGSN) is an 83 kDa actin-binding protein involved in cytoskeletal remodeling, inflammation, and host defense. Its clinical relevance as a biomarker and potential therapeutic agent, particularly in conditions like sepsis, acute respiratory distress syndrome (ARDS), and cystic fibrosis, has driven interest in scalable recombinant expression. However, high-yield production of functionally active gelsolin is hindered by its complex structure and folding requirements. To address this, we developed a scalable, high-yield bacterial expression system that achieves among the highest reported levels of functional recombinant human gelsolin (rGelsolin) using a GST-fusion strategy incorporating a tobacco etch virus (TEV) protease cleavage site, optimized for solubility and downstream processing. High-density fed-batch fermentation in E. coli yielded 5.0 g/L of soluble protein. Following a three-step purification process with removal of the GST tag, 2.1 g/L of tag-free, high-purity rGelsolin with >95 % purity was obtained. Structural characterization by circular dichroism spectroscopy confirmed that rGelsolin adopted a native-like secondary structure and exhibited thermal stability (Tm ∼59 °C). Correct processing of the recombinant protein was verified by N- and C-terminal sequencing. Functional assays demonstrated that rGelsolin bound to and severed actin filaments in a calcium-dependent manner, similar to native plasma gelsolin. These findings demonstrate a scalable, cost-effective platform for producing bioactive rGelsolin in E. coli, with structural and functional features comparable to native pGSN, supporting its potential utility in diagnostic, therapeutic, and structural applications in the context of acute and chronic inflammatory diseases.

人血浆凝胶蛋白（pGSN）是一种83 kDa的肌动蛋白结合蛋白，参与细胞骨架重塑、炎症和宿主防御。它作为一种生物标志物和潜在治疗剂的临床意义，特别是在脓毒症、急性呼吸窘迫综合征（ARDS）和囊性纤维化等疾病中，已经引起了人们对可扩展重组表达的兴趣。然而，功能活性凝胶的高收率生产受到其复杂结构和折叠要求的阻碍。为了解决这个问题，我们开发了一个可扩展的、高产的细菌表达系统，该系统使用gst融合策略，结合烟草蚀刻病毒（TEV）蛋白酶裂解位点，优化了溶解度和下游加工，实现了最高水平的功能性重组人gelsolin （rGelsolin）。高密度补料分批发酵大肠杆菌可获得5.0 g/L可溶性蛋白。经过去除GST标签的三步纯化过程，得到2.1 g/L无标签的高纯度rGelsolin，纯度为bb0 95%。圆二色光谱的结构表征证实了rGelsolin采用类似天然的二级结构，并表现出热稳定性（Tm ~ 59°C）。通过N端和c端测序验证了重组蛋白的正确处理。功能分析表明，rGelsolin以钙依赖的方式结合并切断肌动蛋白丝，类似于天然血浆gelsolin。这些发现证明了在大肠杆菌中生产生物活性rGelsolin的一个可扩展的、具有成本效益的平台，其结构和功能特征与天然pGSN相当，支持其在急慢性炎症性疾病的诊断、治疗和结构应用中的潜在效用。

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引用次数: 0

Validation of cold shock-based technique for purification of recombinant C. elegans Phosphodiesterase 3 protein expressed in E. coli 冷冲击技术纯化重组秀丽隐杆线虫磷酸二酯酶3蛋白的研究。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-07-25 DOI: 10.1016/j.pep.2025.106769

Swapna G. Naik , Dong Keun Rhee , Steven Hockman , Faiyaz Ahmad Khan , Arun Samidurai , Babak Sabouri , Noel Carter , Vincent C. Manganiello

Phosphodiesterase 3 enzymes (PDE3) play important roles in the regulation of adipocyte lipolysis and cardiac contractility by hydrolyzing cAMP and cGMP. This study reports the cloning, expression, and purification of recombinant Caenorhabditis elegans phosphodiesterase 3 (CEPDE3) isoforms, using a cold shock-based technique. The two closely related isoforms of the CEPDE3 gene (isoform F and isoform A) were cloned into the pGEX-6P-1 vector and expressed in E. coli as fusion proteins with a glutathione-S transferase (GST) tag at their amino terminus and purified by affinity chromatography using a glutathione Sepharose column. To optimize expression and recovery of soluble CEPDE3 protein from E. coli, we applied a “cold shock” technique at 4 °C following IPTG induction. Our findings suggest improved protein expression using an N-terminal GST tag instead of a C-terminal 6-histidine (6His) tag. Exposure of GST-tagged CEPDE3 to cold shock improved the protein solubility of CEPDE3 isoforms recovered by affinity chromatography. Studying CEPDE3 expression in C. elegans may enable us to understand the structure-function relationship and help crystallize the proteins to identify its catalytic pocket, contributing to the design of more effective small modulators of PDE3 catalytic activity.

磷酸二酯酶3 （PDE3）通过水解cAMP和cGMP，在脂肪细胞脂解和心脏收缩性调节中发挥重要作用。本研究报道了利用冷冲击技术克隆、表达和纯化重组秀丽隐杆线虫磷酸二酯酶3 （CEPDE3）亚型。将两个密切相关的CEPDE3基因异构体（F异构体和A异构体）克隆到pGEX-6P-1载体中，在大肠杆菌中以融合蛋白的形式表达，在其氨基端带有谷胱甘肽- s转移酶（GST）标签，并使用谷胱甘肽Sepharose柱亲和层析纯化。为了优化大肠杆菌中可溶性CEPDE3蛋白的表达和回收率，我们在IPTG诱导下，在4°C下采用“冷休克”技术。我们的研究结果表明，使用n端GST标签代替c端6-组氨酸（6His）标签可以改善蛋白表达。将gst标记的CEPDE3暴露于冷休克中，通过亲和色谱法回收的CEPDE3亚型的蛋白质溶解度提高。研究CEPDE3在秀丽隐杆线虫中的表达，可以帮助我们了解其结构-功能关系，并有助于蛋白质的结晶鉴定其催化口袋，有助于设计更有效的PDE3催化活性小调节剂。

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引用次数: 0

Novel (R)-Hydroxynitrile lyase enzyme of Pyrus communis: Purification and characterization of its physicochemical and kinetic properties 新型梨(R)-羟基腈裂解酶的纯化及其理化和动力学性质的表征。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-07-23 DOI: 10.1016/j.pep.2025.106785

Asha Kumari , Sheetal , Savitri , Monica Sharma

Hydroxynitrile lyases (HNLs) play a vital role in the asymmetric synthesis of drug precursors and plant defence. In this study, an R-specific HNL (PycHNL) was isolated and purified from Pyrus communis (pear) seeds using ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, achieving a 14.31 % yield and 6.9-fold purification. Native PAGE estimated a molecular mass of ∼92 kDa, and SDS-PAGE revealed heterodimeric subunits of 52 and 39 kDa. High-Performance Liquid Chromatography confirmed the presence of flavin adenine dinucleotide (FAD). Peptide sequencing showed no significant similarity with other Rosaceae HNLs but indicated partial identity with serine carboxypeptidases and α/β hydrolase fold proteins from Arabidopsis species. Optimal enzyme activity was observed at pH 5.5 and 30 °C, with stability for up to 6 h. Kinetic analysis revealed a K_m of 11.75 mM, V_max of 227.27 μmol/min/mg, k_cat of 101.46/min, and a half-life of ∼1.9 days. Chiral HPLC analysis demonstrated that PycHNL preferentially synthesized (R)-mandelonitrile with 96.33 % enantiomeric excess and 86.83 % molar conversion, indicating its potential for biocatalytic applications in producing enantiopure nitriles.

羟基腈裂解酶（HNLs）在药物前体的不对称合成和植物防御中发挥着重要作用。本研究采用硫酸铵沉淀、凝胶过滤、离子交换层析等方法从梨种子中分离纯化了一种r特异性HNL (PycHNL)，产率为14.31%，纯化率为6.9倍。Native PAGE估计分子质量为~ 92 kDa， SDS-PAGE显示异二聚体亚基为52和39 kDa。高效液相色谱法证实黄素腺嘌呤二核苷酸（FAD）的存在。肽段测序结果显示，与其他蔷薇科hnl没有显著的相似性，但与拟南芥的丝氨酸羧肽酶和α/β水解酶折叠蛋白部分相同。在pH 5.5和30°C条件下，酶活性最佳，稳定性可达6小时。动力学分析表明，其Km为11.75 mM， Vmax为227.27 μmol/min/mg， kcat为101.46/min，半衰期为~ 1.9天。手性高效液相色谱分析表明，PycHNL以96.33%的对映体过量和86.83%的摩尔转化率优先合成(R)-mandelonitrile，表明其在制备对映纯腈方面具有潜在的生物催化应用前景。

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引用次数: 0