Protein expression and purification最新文献_第4页

Codon harmonization for improve heterologous expression of human aromatase (CYP19A1) in E. coli cells 密码子协调提高人芳香化酶（CYP19A1）在大肠杆菌细胞中的异种表达

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-25 DOI: 10.1016/j.pep.2025.106864

Marina I. Shaladonova, Yaraslau U. Dzichenka, Veronika V. Shchur, Antos B. Sachanka, Sergey A. Usanov

CYP19A1 (aromatase) is the key enzyme taking part in the process of the conversion of androgens to estrogens thus considering as a major target for treatment of certain types of tumors, especially hormone dependent breast cancer. The purpose of this study is to achieve human aromatase (CYP19A1) from harmonized sequence and identify structural significance of rare codons, involved in the process of codon harmonization. There are several examples of influence of codon harmonization on the protein production and its activity, but these researches didn't include studying any human CYPs, which are difficult-to-produce membrane proteins. The aim of our study is to combine methods of codon harmonization, usage of optimal expression system and conditions, and modifications to the standard purification protocol in order to increase the yield of purified human recombinant aromatase for further usage of the enzyme for the screening of new inhibitors. In our research the structural significance of rare codons for the formation of the secondary structure is revealed, which is implemented in the algorithm of codon harmonization in human aromatase sequence. The combined approach with codon harmonization technique and one-step purification enables to achieve over 300 nmol of homogeneous, catalytically active human CYP19A1 per Liter of culture. We expect the results obtained will provide valuable information for protein engineering and enzymology.

CYP19A1（芳香化酶）是参与雄激素向雌激素转化过程的关键酶，因此被认为是治疗某些类型肿瘤的主要靶点，尤其是激素依赖性乳腺癌。本研究的目的是从协调序列中获得人芳香化酶（CYP19A1），并鉴定参与密码子协调过程的罕见密码子的结构意义。密码子协调对蛋白质产生及其活性的影响有几个例子，但这些研究都没有包括对人类cyp的研究，cyp是一种难以产生的膜蛋白。本研究的目的是结合密码子协调方法、最佳表达系统和条件的使用以及对标准纯化方案的修改，以提高纯化的重组人芳香酶的产量，从而进一步利用该酶筛选新的抑制剂。本研究揭示了稀有密码子对二级结构形成的结构意义，并将其应用于人类芳香酶序列密码子协调算法中。结合密码子协调技术和一步纯化，每升培养物可获得超过300 nmol的均匀、催化活性的人CYP19A1。本研究结果将为蛋白质工程和酶学研究提供有价值的信息。

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引用次数: 0

Systematic optimization for high-level secretory expression of a thermostable zearalenone hydrolase ZENM in Pichia pastoris 耐热玉米赤霉烯酮水解酶ZENM在毕赤酵母中高水平分泌表达的系统优化

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-25 DOI: 10.1016/j.pep.2025.106843

Shida Zhao , Qin Zhou , Yuzhuo Wu , Ying Hou , Qi Wu , Ding Li , Jianhong Xu

Zearalenone (ZEN), a mycotoxin commonly found in cereal grains, poses a significant threat to livestock health. ZEN hydrolase is considered a key tool for detoxifying ZEN contamination due to its high efficiency, cost-effectiveness, and ability to completely degrade the toxin. Among these enzymes, ZEN hydrolase ZENM is a promising candidate for industrial applications owing to its superior thermostability. In this study, ZENM was heterologously expressed in the Pichia pastoris system. The enzyme exhibited optimal activity at pH 8.0 and 60 °C, and maintained over 90 % of its relative activity within the 50–60 °C range. Its kinetic parameters, the apparent Michaelis-Menten constant (K_m, _app) and maximal reaction velocity (V_max), were determined to be 21.59 ± 7.34 μM and 0.031 ± 0.002 μM s⁻¹ mg⁻¹, respectively. The yield of ZENM was increased from 6.70 U/mL to 41.55 U/mL by utilizing the pre-Ost1-pro-α-factor signal peptide. Increasing the gene dosage further enhanced the yield to 50.87 U/mL. Furthermore, co-expression with molecular chaperones PDI and SEC12 elevated the yield to 72.29 U/mL and 68.88 U/mL, respectively. Ultimately, a 10.79-fold increase in ZENM production was achieved in shake flask cultures compared to the initial strain. This research establishes a solid foundation for advancing the industrial application of ZENM.

玉米赤霉烯酮（ZEN）是谷物中常见的一种霉菌毒素，对牲畜健康构成重大威胁。ZEN水解酶被认为是解毒ZEN污染的关键工具，因为它具有高效率，成本效益和完全降解毒素的能力。在这些酶中，ZEN水解酶因其优异的热稳定性而成为工业应用的有前途的候选酶。在本研究中，ZENM在毕赤酵母系统中异种表达。该酶在pH 8.0和60℃条件下表现出最佳活性，在50 ~ 60℃范围内保持90%以上的相对活性。测定其表观Michaelis-Menten常数（Km, app）和最大反应速度（Vmax）分别为21.59±7.34 μM和0.031±0.002 μM s−1 mg−1。利用前ost1 -pro-α-因子信号肽可将ZENM的产率从6.70 U/mL提高到41.55 U/mL。增加基因投加量可进一步提高产率至50.87 U/mL。与分子伴侣蛋白PDI和SEC12共表达后，产量分别提高到72.29 U/mL和68.88 U/mL。最终，与初始菌株相比，摇瓶培养的ZENM产量增加了10.79倍。本研究为推进ZENM的工业应用奠定了坚实的基础。

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引用次数: 0

Synergistic effect of LTB and Mn2+ adjuvants on immunogenicity of multi-epitope peptides from Staphylococcus aureus LTB和Mn2+佐剂对金黄色葡萄球菌多表位肽免疫原性的协同作用

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-24 DOI: 10.1016/j.pep.2025.106862

Jinzhu Ma , Shuyu Wei , Ming Xu , Changquan Liu , Mingyang Tao , Youxi Fu , Kexin Liu , Siqi Wang , Simiao Yu , Liquan Yu , Beiyan Wang , Baifen Song

Here, a novel combined adjuvant of heat-labile enterotoxin B (LTB) as an intramolecular adjuvant plus Mn²⁺ was prepared to enhance the immunogenicity of multi-epitope (MEP) peptides derived from Staphylococcus aureus (S. aureus). Initially, the mep and ltb-mep fragments were amplified and inserted into pET-28a vectors, respectively, to generate the recombinant plasmids pET28a-mep and pET28a-ltb-mep. Then, the expression of MEP and LTB-MEP was verified by Western blot. Subsequently, the MEP and LTB-MEP proteins were subjected to bioinformatics analysis. Kunming mice were immunized with the LTB-MEP proteins plus Mn²⁺ adjuvants. After the second immunization, the mice immunized with LTB-MEP plus Mn²⁺ exhibited significantly enhanced splenic lymphocyte proliferation, increased secretion of IFN-β, IFN-γ, IL-2, IL-6, and IL-17 by splenic lymphocytes, and significantly enhanced the generation of antibodies against MEP in serum, and improved survival rate following S. aureus challenge. The data showed that the novel LTB-Mn²⁺ combined adjuvant exerted a synergistic effect, significantly enhanced the immunogenicity of MEP, and provided a new strategy for improving the efficacy of vaccines against S. aureus.

本研究制备了一种由热不稳定肠毒素B （LTB）与Mn2+作为分子内佐剂的新型联合佐剂，以增强来自金黄色葡萄球菌（S. aureus）的多表位（MEP）肽的免疫原性。首先扩增mep和ltb-mep片段，分别插入pET-28a载体中，生成重组质粒pET28a-mep和pET28a-ltb-mep。Western blot检测MEP和LTB-MEP的表达。随后，对MEP和LTB-MEP蛋白进行生物信息学分析。用LTB-MEP蛋白加Mn2+佐剂免疫昆明小鼠。第二次免疫后，经LTB-MEP + Mn2+免疫的小鼠脾脏淋巴细胞增殖明显增强，脾脏淋巴细胞分泌IFN-β、IFN-γ、IL-2、IL-6和IL-17增加，血清中MEP抗体的产生显著增加，金黄色葡萄球菌攻毒后存活率提高。结果表明，新型LTB-Mn2+联合佐剂发挥了协同作用，显著增强了MEP的免疫原性，为提高金黄色葡萄球菌疫苗的疗效提供了新的策略。

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引用次数: 0

Expression, purification, and biophysical characterization of liquid-liquid phase separation of full-length hnRNPA2B1 hnRNPA2B1全长蛋白的表达、纯化及液-液分离生物物理特性研究

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-24 DOI: 10.1016/j.pep.2025.106861

Luis Fernando Durán-Armenta , Attila Meszaros , Julia Malo Pueyo , Steven Janvier , Tamas Lazar , Dominique Maes , Remy Loris , Peter Tompa

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is a multifunctional RNA-binding protein involved in RNA maturation and mRNA transport. It has recently been shown to undergo liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles. Moreover, dysregulation of LLPS is associated with the formation of pathogenic protein aggregates, in which hnRNPA2B1 is frequently found. Despite its biological and pathological relevance, studies on the full-length protein remain limited due to its intrinsically disordered low-complexity domain, which renders hnRNPA2B1 highly aggregation-prone and difficult to purify. In this study, we report the successful expression and purification of full-length hnRNPA2B1 with high purity and minimal nucleic acid contamination. By optimizing buffer conditions, specifically ionic strength and pH, we maintained the protein in solution following cleavage of its solubility tag. Preliminary in vitro characterization under near-physiological conditions reveals that purified hnRNPA2B1 undergoes LLPS, forming dynamic liquid-like droplets that grow and mature into amorphous aggregates. Our approach provides a robust method for purifying hnRNPA2B1 suitable for LLPS and aggregation studies. This strategy may also be useful to purify other aggregation-prone, intrinsically disordered proteins.

异质核核糖核蛋白A2/B1 （hnRNPA2B1）是一种参与RNA成熟和mRNA转运的多功能RNA结合蛋白。它最近被证明经历液-液相分离（LLPS），有助于无膜细胞器的组装。此外，LLPS的失调与致病性蛋白聚集体的形成有关，其中经常发现hnRNPA2B1。尽管hnRNPA2B1具有生物学和病理学意义，但由于其内在无序的低复杂性结构域，对其全长蛋白的研究仍然有限，这使得hnRNPA2B1极易聚集且难以纯化。在这项研究中，我们成功地表达和纯化了全长hnRNPA2B1，具有高纯度和最小的核酸污染。通过优化缓冲条件，特别是离子强度和pH值，我们将蛋白质在溶解性标签裂解后保持在溶液中。在近生理条件下的初步体外表征表明，纯化的hnRNPA2B1经历了LLPS，形成了动态的液体状液滴，并生长成熟为无定形聚集体。我们的方法提供了一种强大的纯化hnRNPA2B1的方法，适用于LLPS和聚集研究。这种方法也可用于纯化其他容易聚集的、内在无序的蛋白质。

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引用次数: 0

Purification of antifreeze proteins from tussah silkworm (Antheraea pernyi) via polyethylene glycol precipitation coupled with aqueous two-phase extraction 聚乙二醇沉淀法-双水相萃取法纯化柞蚕抗冻蛋白。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-21 DOI: 10.1016/j.pep.2025.106856

Xinhui Liu, Yiqun Yin, Xin Xu, Tingting Tan, Taoyu Zhou, Yan Wang

Antifreeze proteins (AFPs) represent specialized proteins capable of inhibiting ice crystal growth and modifying ice crystal morphology. They hold significant potential in biopharmaceuticals, food storage, and cryopreservation. As a characteristic biological resource in China, the tussah silkworm (Antheraea pernyi) is both readily available and rich in AFPs. However, there is currently no efficient, mild, and scalable method for separating and purifying these AFPs. To that end, we adopted a polyethylene glycol (PEG)-based purification strategy coupled with aqueous two-phase extraction (ATPE) to efficiently isolate AFPs from tussah silkworms. First, PEG was used to precipitate the crude extract, thus removing contaminating proteins. The supernatant was then subjected to phase separation by adding saturated ammonium sulfate solution, during which the AFPs were primarily enriched in the upper phase. Through single-factor experiments, we determined the optimal conditions: a PEG molecular weight of 20,000, a PEG concentration of 8 % (w/w), and an ammonium sulfate concentration of 26 % (w/w). Under these conditions, an extraction efficiency of 79.75 ± 5.29 % (n = 3) was achieved. At an AFP concentration of 68.65 mg/mL, the thermal hysteresis activity was 1.82 ± 0.03 °C (n = 3). To confirm the ice-shaping activity, we analyzed the purified product using a nanoliter osmometer, which exhibited hexagonal ice crystals. The target protein was obtained with a purity of 72.56 ± 2.20 % (n = 3). SDS-PAGE analysis showed a single band at 75 kDa, confirming that the protein was substantially enriched. Cytotoxicity assays demonstrated that the extracted AFP exhibited excellent biocompatibility. The coupling of PEG precipitation and ATPE proved effective in rapidly enriching the target protein and removing impurities. This approach provides an application of a potentially scalable strategy for large-scale AFP preparation from tussah silkworm.

抗冻蛋白（AFPs）是一种能够抑制冰晶生长和改变冰晶形态的特殊蛋白。它们在生物制药、食品储存和低温保存方面具有巨大的潜力。柞蚕（Antheraea pernyi）是一种易得且富含抗菌肽的中国特色生物资源。然而，目前还没有有效的、温和的、可扩展的方法来分离和纯化这些afp。为此，我们采用聚乙二醇（PEG）为基础的纯化策略，结合水两相萃取（ATPE），有效地分离柞蚕的AFPs。首先，用聚乙二醇沉淀粗提取物，从而去除污染的蛋白质。然后加入饱和硫酸铵溶液对上清液进行相分离，在此过程中，AFPs主要富集于上相。通过单因素实验，我们确定了最佳条件：PEG分子量为20,000，PEG浓度为8% (w/w)，硫酸铵浓度为26% （w/w）。在此条件下，提取效率为79.75±5.29% （n = 3）。在AFP浓度为68.65 mg/mL时，热滞活性为1.82±0.03°C （n = 3）。为了确认冰的形成活性，我们用纳升渗透仪分析了纯化后的产品，显示出六边形冰晶。得到的目标蛋白纯度为72.56±2.20% （n = 3）。SDS-PAGE分析显示在75 kDa处有一条条带，证实该蛋白大量富集。细胞毒性实验表明，提取的AFP具有良好的生物相容性。PEG沉淀与ATPE的偶联被证明可以快速富集目标蛋白并去除杂质。该方法为柞蚕AFP的大规模制备提供了一种潜在的可扩展策略。

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引用次数: 0

Bench-top NMR of water signals: A non-destructive tool for biomacromolecule characterization 水信号的台式核磁共振：生物大分子表征的非破坏性工具。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-19 DOI: 10.1016/j.pep.2025.106855

Tomoto Ura , Taiji Oyama , Chiaki Nishimura

Bench-top NMR spectroscopy has emerged as an accessible, non-destructive tool for analyzing biomacromolecules in solution. This review focuses on how water proton NMR (wNMR) provides sensitive readouts of protein states in solution. We highlight representative studies on monoclonal antibodies, insulin, and vaccines under diverse stresses, where wNMR measurements consistently correlate with aggregation and stability outcomes. In parallel, we revisit the fundamental principles of wNMR, focusing on the molecular origins of water relaxation and the roles of hydration dynamics, proton exchange, and water confinement near protein surfaces. To illustrate these mechanisms, we present simple illustrative experiments using bench-top wNMR and ATR-FTIR spectroscopy with aqueous salts and polymers, revealing how non-protein solutes modulate hydrogen-bond networks and water dynamics. Finally, we discuss emerging perspectives that extend the scope of bench-top NMR beyond protein formulations. Together, these developments position bench-top NMR not only as a practical tool for stability assessment but also as a promising framework for probing water-mediated phenomena across biopharmaceutical and soft-matter systems.

台式核磁共振波谱已成为分析溶液中生物大分子的一种方便、非破坏性的工具。本文综述了水质子核磁共振（wNMR）如何提供溶液中蛋白质状态的敏感读数。我们重点介绍了在不同压力下对单克隆抗体、胰岛素和疫苗的代表性研究，其中wNMR测量结果与聚集性和稳定性结果一致相关。同时，我们回顾了wNMR的基本原理，重点关注水弛豫的分子起源以及水合动力学、质子交换和蛋白质表面附近的水约束的作用。为了说明这些机制，我们使用wNMR和ATR-FTIR光谱对含水盐和聚合物进行了简单的说明性实验，揭示了非蛋白溶质如何调节氢键网络和水动力学。最后，我们讨论了将台式核磁共振范围扩展到蛋白质配方之外的新兴观点。总之，这些发展使台式核磁共振不仅作为稳定性评估的实用工具，而且作为探测生物制药和软物质系统中水介导现象的有前途的框架。

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引用次数: 0

The advantage of high pH eluting Protein A resins over their regular counterparts in aggregate and host cell protein clearance 高pH洗脱蛋白A树脂在聚集体和宿主细胞蛋白清除方面的优势。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-17 DOI: 10.1016/j.pep.2025.106859

Ziyang Li , Lixia Hu , Yifeng Li

Protein A affinity chromatography is the most well-established method and gold standard for antibody purification. Typically, low pH (i.e., 3.0–3.5) is required for elution of bound antibodies from a Protein A column. However, for acid-sensitive antibodies, this low pH condition can trigger severe aggregation. Therefore, alternative Protein A resins supporting mild elution are highly desirable. To meet this need, recently Purolite and Cytiva both developed high pH eluting Protein A resins (i.e., Jetted A50 HipH and MabSelect mild elution, respectively). We previously showed that Jetted A50 HipH not only supports mild pH elution but also exhibits improved aggregate separation capability. In the current work, with four case studies, we compared the above-mentioned two high pH eluting Protein A resins with their regular counterparts (i.e., Jetted A50 and MabSelect SuRe LX, respectively) and demonstrated that the two special Protein A resins provided much better aggregate removal and host cell protein (HCP) clearance. Thus, in addition to supporting mild elution, the two high pH eluting Protein A resins also have an edge in terms of impurity clearance.

蛋白A亲和层析是抗体纯化最完善的方法和金标准。通常，从蛋白a柱中洗脱结合抗体需要低pH（即3.0-3.5）。然而，对于酸敏感抗体，这种低pH条件会引发严重的聚集。因此，支持温和洗脱的替代蛋白A树脂是非常可取的。为了满足这一需求，最近Purolite和Cytiva都开发了高pH值洗脱蛋白A树脂（即Jetted A50 HipH和MabSelect温和洗脱）。我们之前的研究表明，Jetted A50 HipH不仅支持温和的pH洗脱，而且还表现出更好的骨料分离能力。在目前的工作中，通过四个案例研究，我们将上述两种高pH洗脱蛋白A树脂与常规树脂（分别为Jetted A50和MabSelect SuRe LX）进行了比较，并证明这两种特殊的蛋白A树脂可以更好地去除聚集体和清除宿主细胞蛋白（HCP）。因此，除了支持温和洗脱外，两种高pH洗脱蛋白A树脂在杂质清除方面也具有优势。

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引用次数: 0

Strategic truncation improves recombinant expression of buffalo Fibulin-5 (FBLN5) in Escherichia coli 策略截断提高水牛纤维蛋白-5 （FBLN5）在大肠杆菌中的重组表达

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-17 DOI: 10.1016/j.pep.2025.106858

Deviyani Mahajan , Neha Sarova , Jyoti Rustagi , Shwetali Prajapati , Jai Kumar Kaushik , Ashok Kumar Mohanty , Manoj Kumar Jena , Sudarshan Kumar

Background

Accurate early pregnancy detection is crucial for enhancing herd productivity in bovines. There is growing interest in identifying reliable protein biomarkers that can be used to develop rapid, sensitive, and cost-effective diagnostic tools. This study focused on buffalo fibulin-5 (FBLN5), an extracellular matrix glycoprotein involved in tissue remodeling and cell adhesion, and a potential early pregnancy biomarker.

Methods

In this study, we used the pET-32a(+) expression vector and three E. coli expression hosts. Heat-shock transformation was performed in chemically competent cells (CaCl₂ method), and the expression was optimized at 18 °C and 37 °C. Recombinant truncated FBLN5 was purified through immobilized metal affinity chromatography (IMAC) on nickel resin and subsequently characterized by SDS-PAGE and Western blotting.

Results

Optimization of various expression conditions, such as temperature, induction, incubation time, and additives, did not result in any detectable expression of full-length FBLN5. Therefore, a truncated version of FBLN5 was designed based on the predicted antigenic regions to increase its immunogenicity and simplify its recombinant expression. Truncated FBLN5 (Trx-tagged and 6 × His-tagged) expressed well in BL21Codon Plus (DE3)-RIL and Lemo21(DE3) at 18 °C with 1 mM IPTG induction. Furthermore, we successfully purified soluble truncated FBLN5 and confirmed its molecular weight (∼36 kDa) and identity using western blotting with enhanced chemiluminescence (ECL).

Conclusion

Truncation of full-length FBLN5 resulted in successful cloning, expression, and purification of this otherwise difficult to express protein. This study provides a solid foundation for future immunological studies aimed to evaluate FBLN5 potential as diagnostic marker in early pregnancy detection in bovine species.

背景：准确的早期妊娠检测对提高牛群生产力至关重要。人们对鉴定可靠的蛋白质生物标志物越来越感兴趣，这些标志物可用于开发快速、敏感和具有成本效益的诊断工具。这项研究的重点是水牛纤维蛋白-5 (FBLN5)，这是一种参与组织重塑和细胞粘附的细胞外基质糖蛋白，也是一种潜在的早期妊娠生物标志物。方法：本研究采用pET-32a（+）表达载体和3个大肠杆菌表达宿主。热冲击转化为化学活性细胞（CaCl2法），在18°C和37°C条件下表达最佳。重组截断FBLN5通过镍树脂固定化金属亲和层析（IMAC）纯化，并通过SDS-PAGE和Western blotting对其进行表征。结果：优化各种表达条件，如温度、诱导、孵育时间、添加剂等，均未检测到FBLN5全长的表达。因此，我们根据预测的抗原区域设计了截断版FBLN5，以提高其免疫原性，简化其重组表达。截断的FBLN5 （trx标记和6×His-tagged）在18°C、1 mM IPTG诱导下在BL21Codon Plus (DE3)-RIL和Lemo21（DE3）中表达良好。此外，我们成功地纯化了可溶性截断的FBLN5，并使用增强化学发光（ECL）的western blotting证实了其分子量（~ 36 kDa）和身份。结论：截断全长FBLN5，成功克隆、表达和纯化了这种难以表达的蛋白。本研究为进一步开展FBLN5作为牛早期妊娠诊断标志物的免疫学研究奠定了基础。

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引用次数: 0

Extraction and characterization of functional duckweed proteins using alkaline, enzymatic, and ultrasound-assisted techniques 利用碱性、酶和超声辅助技术提取和表征功能性浮萍蛋白。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-17 DOI: 10.1016/j.pep.2025.106860

Ayça Akyüz, Seda Ersus

The urgent need for sustainable, plant-based protein sources has brought aquatic crops like duckweed (Lemnaceae) into focus due to their high protein content, fast growth, and minimal resource requirements. This study aims to optimize and compare alkaline, enzyme-assisted, and ultrasound-assisted extraction techniques to produce high-yield, functional protein concentrates from Lemna minor. Key extraction parameters—pH, temperature, and solvent-to-solid ratio—were systematically optimized, identifying pH 9, 55 °C, and 5 mL/g as ideal conditions. Ultrasound treatment (100 % amplitude, 10 min) and Alcalase L enzyme application significantly enhanced protein yields. Ultrasound-assisted extraction proved most effective, increasing protein yield by more than fivefold compared to conventional alkaline methods, reaching 60.09 % protein content on a dry basis. The resulting protein concentrates displayed desirable functional properties, including excellent solubility (92.19 % at pH 9), foaming capacity (92.62 %), and emulsion activity, all of which are critical for food applications. Comprehensive structural and compositional analyses confirmed the presence of essential amino acids, high mineral content (notably calcium and phosphorus), and acceptable techno-functional behavior. SEM and FTIR analyses supported the structural integrity of the extracted proteins, while flowability assessments suggested suitability for powder-based formulations. This research demonstrates that ultrasound-assisted protein isolation from duckweed offers a scalable and efficient strategy for producing high-quality protein concentrates. These findings position duckweed as a promising, sustainable protein source with strong potential for incorporation into functional foods and novel plant-based formulations.

对可持续的植物性蛋白质来源的迫切需求使浮萍（Lemnaceae）等水生作物成为人们关注的焦点，因为它们蛋白质含量高，生长快，资源需求少。本研究旨在优化和比较碱性、酶辅助和超声辅助提取技术，以获得高产量、功能性的苦荬菜浓缩蛋白。系统优化了关键提取参数pH、温度和液固比，确定pH 9、55℃和5 mL/g为理想条件。超声处理（100%振幅，10分钟）和Alcalase L酶的应用显著提高了蛋白质产量。超声辅助提取被证明是最有效的，与传统的碱性方法相比，蛋白质产量提高了5倍以上，在干燥的基础上达到60.09%的蛋白质含量。所得到的蛋白质浓缩物显示出理想的功能特性，包括优异的溶解度（pH值9时为92.19%）、起泡能力（92.62%）和乳液活性，所有这些都是食品应用的关键。全面的结构和成分分析证实了必需氨基酸的存在，高矿物质含量（特别是钙和磷），以及可接受的技术功能行为。SEM和FTIR分析支持了提取蛋白质的结构完整性，而流动性评估表明粉末基配方的适用性。本研究表明，超声辅助从浮萍中分离蛋白质提供了一种可扩展和有效的生产高质量浓缩蛋白的策略。这些发现表明浮萍是一种有前途的、可持续的蛋白质来源，具有很强的潜力，可以被纳入功能性食品和新型植物性配方中。

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引用次数: 0

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