Protein expression and purification最新文献_第4页

Solubilization and refolding of inclusion bodies by detergents. 洗涤剂对包裹体的增溶和再折叠作用。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-12-01 Epub Date: 2025-08-07 DOI: 10.1016/j.pep.2025.106791

Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto

Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.

重组蛋白在生物试剂和生物制药的开发中发挥着重要作用。在这里，我们将主要回顾重组蛋白在包涵体中表达时的重折叠，尽管增强可溶性表达的策略被描述为重折叠包涵体的替代方法。这些策略包括，但不限于，在细胞培养基中添加化学伴侣，修改细胞裂解缓冲液和使用提高溶解度的融合标签。另一种增强溶解度的方法是为膜蛋白生成脂质复合物，形成不溶性的无脂蛋白。在各种增溶和再折叠技术中，介绍了使用变性剂、碱性pH值和压力的增溶和再折叠技术，重点介绍了使用洗涤剂的增溶和再折叠技术，这是一种有效且经济的技术。综述了十二烷基硫酸钠、月桂酰谷氨酸、萨科齐和十六烷基三甲基铵的广泛应用。缓慢或逐步去除用于增溶的变性剂或离子洗涤剂似乎在成功的再折叠中发挥关键作用，通过在再折叠过程中保持蛋白质的溶解度。在碱性再折叠中，缓慢的pH调节也有助于维持蛋白质的溶解度。加压折纸时，少量盐酸胍辅助折纸。

{"title":"Solubilization and refolding of inclusion bodies by detergents.","authors":"Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto","doi":"10.1016/j.pep.2025.106791","DOIUrl":"10.1016/j.pep.2025.106791","url":null,"abstract":"<p><p>Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106791"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812236","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

Analysis of the antibacterial and anti-inflammatory functions of bovine lactoferrin functional fragment expressed in Escherichia coli via engineering 大肠杆菌表达的牛乳铁蛋白功能片段抑菌抗炎功能的工程分析。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-29 DOI: 10.1016/j.pep.2025.106865

Hui Teng, Zizi Ding, Yujie Wang, Xueliang Liu, Xiangshan Tang, Xuewen Zhang

The bovine lactoferrin is a natural iron-binding glycoprotein that has attracted widespread attention due to its functions such as antibacterial, antiviral, anti-tumor, anti-inflammatory and immunomodulatory effects. In this study, the lactoferrin functional fragment (BlfFf) was expressed in Escherichia coli by genetic engineering expression. The expressed BlfFf mainly existed as inclusion bodies in the host cells. After collecting the inclusion bodies, the BlfFf purified polypeptide was obtained through dissolution, renaturation and further chromatography preparation. The antibacterial test of the purified BlfFf indicated that BlfFf did not exhibit antibacterial activity before pepsin hydrolysis. After hydrolysis by pepsin, BlfFf has obvious antibacterial properties against both Staphylococcus aureus and Escherichia coli represent Gram-positive and Gram-negative bacteria each. Cell experiments have demonstrated that the prepared BlfFf can significantly reduce the expression of inflammation-related genes in the cell model of LPS-stimulated macrophages (Raw264.7) with inflammation model, showing a significant inflammatory inhibitory effect. In Raw264.7 cells treated with BlfFf, the expression levels of various pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and iNOS were significantly decreased. Lower concentrations of BlfFf show a certain cell-promoting growth effect. This experiment demonstrated the multiple biological activities and functions of bacterial genetic engineering in expressing bovine lactoferrin peptides.

牛乳铁蛋白是一种天然的铁结合糖蛋白，因其具有抗菌、抗病毒、抗肿瘤、抗炎和免疫调节等功能而受到广泛关注。本研究利用基因工程技术在大肠杆菌中表达乳铁蛋白功能片段（BlfFf）。表达的BlfFf主要以包涵体形式存在于宿主细胞中。收集包涵体后，通过溶出、复性和进一步层析制备得到BlfFf纯化多肽。对纯化的BlfFf进行抑菌实验表明，在胃蛋白酶水解前BlfFf不表现出抑菌活性。经胃蛋白酶水解后，BlfFf对代表革兰氏阳性菌和革兰氏阴性菌的金黄色葡萄球菌和大肠杆菌均有明显的抑菌作用。细胞实验表明，制备的BlfFf可以显著降低lps刺激的巨噬细胞（Raw264.7）炎症模型中炎症相关基因的表达，显示出明显的炎症抑制作用。在经BlfFf处理的Raw264.7细胞中，各种促炎细胞因子IL-1β、TNF-α、IL-6和iNOS的表达水平均显著降低。低浓度BlfFf具有一定的促细胞生长作用。本实验验证了细菌基因工程表达牛乳铁蛋白多肽的多种生物学活性和功能。

{"title":"Analysis of the antibacterial and anti-inflammatory functions of bovine lactoferrin functional fragment expressed in Escherichia coli via engineering","authors":"Hui Teng, Zizi Ding, Yujie Wang, Xueliang Liu, Xiangshan Tang, Xuewen Zhang","doi":"10.1016/j.pep.2025.106865","DOIUrl":"10.1016/j.pep.2025.106865","url":null,"abstract":"<div><div>The bovine lactoferrin is a natural iron-binding glycoprotein that has attracted widespread attention due to its functions such as antibacterial, antiviral, anti-tumor, anti-inflammatory and immunomodulatory effects. In this study, the lactoferrin functional fragment (BlfFf) was expressed in <em>Escherichia coli</em> by genetic engineering expression. The expressed BlfFf mainly existed as inclusion bodies in the host cells. After collecting the inclusion bodies, the BlfFf purified polypeptide was obtained through dissolution, renaturation and further chromatography preparation. The antibacterial test of the purified BlfFf indicated that BlfFf did not exhibit antibacterial activity before pepsin hydrolysis. After hydrolysis by pepsin, BlfFf has obvious antibacterial properties against both <em>Staphylococcus aureus</em> and <em>Escherichia coli</em> represent Gram-positive and Gram-negative bacteria each. Cell experiments have demonstrated that the prepared BlfFf can significantly reduce the expression of inflammation-related genes in the cell model of LPS-stimulated macrophages (Raw264.7) with inflammation model, showing a significant inflammatory inhibitory effect. In Raw264.7 cells treated with BlfFf, the expression levels of various pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and iNOS were significantly decreased. Lower concentrations of BlfFf show a certain cell-promoting growth effect. This experiment demonstrated the multiple biological activities and functions of bacterial genetic engineering in expressing bovine lactoferrin peptides.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106865"},"PeriodicalIF":1.2,"publicationDate":"2025-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145654533","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

Characterization of a novel GH5 β-mannanase from Hahella sp. CR1 and its synergistic role in degradation of spent coffee grounds 来自Hahella sp. CR1的新型GH5 β-甘露聚糖酶的表征及其在废咖啡渣降解中的协同作用

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification

Pub Date : 2025-11-29 DOI: 10.1016/j.pep.2025.106863

Melvin Chun Yun Tan, Kok Jun Liew, Faezah Mohd Salleh, Chun Shiong Chong

An efficient enzymatic degradation of plant biomass is essential for agricultural waste valorization and biofuel production. β-Mannanase is a key lignocellulolytic enzyme that targets mannan, a major component in plant cell walls. Hahella spp. are known to produce cellulolytic enzymes, but no β-mannanase was characterized from this genus up to date. In this study, a gene encoding for β-mannanase (HcrMan1) was identified and isolated from the genome of Hahella sp. CR1. HcrMan1 encodes a multidomain enzyme comprising a GH5 subfamily 8 catalytic domain, an immunoglobulin-like (Ig) linker domain, and a CBM2 substrate-binding domain. The presence of the rare Ig-like linker and a structurally distinct CBM2 makes its domain architecture unique compared to homologs. To investigate their contributions in the enzyme reaction, the wild-type HcrMan1 and two truncated variants (HcrMan1ΔCBM2 and HcrMan1ΔIgΔCBM2) were heterologously expressed and purified. Results revealed that the removal of Ig-like linker domain significantly affected protein solubility, whereas the deletion of CBM2 reduced the substrate-binding affinity but enhanced catalytic efficiency (K_cat/K_m) from 18.08 to 22.45. Truncated HcrMan1ΔCBM2 demonstrated a higher specific activity against locust bean gum (LBG) at optimal conditions (50 °C, pH 8.0) compared to its wild type. Furthermore, HcrMan1ΔCBM2 exhibited enhanced enzymatic performance when combined with the commercial cellulase CTec2 in saccharifying spent coffee grounds (SCG), achieving significant increase in reducing sugar release after alkali, ammonia and sonication pretreatment compared to CTec2 alone. These noteworthy findings highlight the compatibility and industrial relevance of engineered HcrMan1 in applications that involved with the utilization of lignocellulosic biomass.

有效的酶降解植物生物质对农业废弃物增值和生物燃料生产至关重要。β-甘露聚糖酶是一种针对甘露聚糖的关键木质纤维素水解酶，甘露聚糖是植物细胞壁的主要成分。已知Hahella属产生纤维素水解酶，但迄今尚未从该属中鉴定出β-甘露聚糖酶。本研究从Hahella sp. CR1基因组中鉴定并分离到一个编码β-甘露聚糖酶（HcrMan1）的基因。HcrMan1编码一种多结构域酶，包括GH5亚家族8催化结构域、免疫球蛋白样（Ig）连接结构域和CBM2底物结合结构域。罕见的类igg连接体和结构独特的CBM2的存在使其结构域结构与同源物相比是独特的。为了研究它们在酶反应中的作用，我们对野生型HcrMan1和两个截短变体（HcrMan1ΔCBM2和HcrMan1ΔIgΔCBM2）进行了异源表达和纯化。结果表明，去除Ig-like连接域显著影响蛋白质的溶解度，而CBM2的缺失降低了底物结合亲和力，但提高了催化效率（Kcat/Km），从18.08提高到22.45。截断的HcrMan1ΔCBM2在最佳条件（50°C, pH 8.0）下对刺槐豆胶（LBG）的比活性高于野生型。此外，HcrMan1ΔCBM2与商用纤维素酶CTec2联合使用时，在将废咖啡渣（SCG）糖化时表现出更强的酶促性能，与单独使用CTec2相比，碱、氨和超声预处理后还原糖的释放量显著增加。这些值得注意的发现突出了工程HcrMan1在涉及木质纤维素生物质利用的应用中的兼容性和工业相关性。

{"title":"Characterization of a novel GH5 β-mannanase from Hahella sp. CR1 and its synergistic role in degradation of spent coffee grounds","authors":"Melvin Chun Yun Tan, Kok Jun Liew, Faezah Mohd Salleh, Chun Shiong Chong","doi":"10.1016/j.pep.2025.106863","DOIUrl":"10.1016/j.pep.2025.106863","url":null,"abstract":"<div><div>An efficient enzymatic degradation of plant biomass is essential for agricultural waste valorization and biofuel production. β-Mannanase is a key lignocellulolytic enzyme that targets mannan, a major component in plant cell walls. <em>Hahella</em> spp. are known to produce cellulolytic enzymes, but no β-mannanase was characterized from this genus up to date. In this study, a gene encoding for β-mannanase (<em>HcrMan1</em>) was identified and isolated from the genome of <em>Hahella</em> sp. CR1. <em>HcrMan1</em> encodes a multidomain enzyme comprising a GH5 subfamily 8 catalytic domain, an immunoglobulin-like (Ig) linker domain, and a CBM2 substrate-binding domain. The presence of the rare Ig-like linker and a structurally distinct CBM2 makes its domain architecture unique compared to homologs. To investigate their contributions in the enzyme reaction, the wild-type HcrMan1 and two truncated variants (HcrMan1ΔCBM2 and HcrMan1ΔIgΔCBM2) were heterologously expressed and purified. Results revealed that the removal of Ig-like linker domain significantly affected protein solubility, whereas the deletion of CBM2 reduced the substrate-binding affinity but enhanced catalytic efficiency (K<sub>cat</sub>/K<sub>m</sub>) from 18.08 to 22.45. Truncated HcrMan1ΔCBM2 demonstrated a higher specific activity against locust bean gum (LBG) at optimal conditions (50 °C, pH 8.0) compared to its wild type. Furthermore, HcrMan1ΔCBM2 exhibited enhanced enzymatic performance when combined with the commercial cellulase CTec2 in saccharifying spent coffee grounds (SCG), achieving significant increase in reducing sugar release after alkali, ammonia and sonication pretreatment compared to CTec2 alone. These noteworthy findings highlight the compatibility and industrial relevance of engineered HcrMan1 in applications that involved with the utilization of lignocellulosic biomass.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106863"},"PeriodicalIF":1.2,"publicationDate":"2025-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145654828","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

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。

{"title":"Codon harmonization for improve heterologous expression of human aromatase (CYP19A1) in E. coli cells","authors":"Marina I. Shaladonova, Yaraslau U. Dzichenka, Veronika V. Shchur, Antos B. Sachanka, Sergey A. Usanov","doi":"10.1016/j.pep.2025.106864","DOIUrl":"10.1016/j.pep.2025.106864","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106864"},"PeriodicalIF":1.2,"publicationDate":"2025-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145615544","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

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。

{"title":"Systematic optimization for high-level secretory expression of a thermostable zearalenone hydrolase ZENM in Pichia pastoris","authors":"Shida Zhao , Qin Zhou , Yuzhuo Wu , Ying Hou , Qi Wu , Ding Li , Jianhong Xu","doi":"10.1016/j.pep.2025.106843","DOIUrl":"10.1016/j.pep.2025.106843","url":null,"abstract":"<div><div>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 <em>Pichia pastoris</em> 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<sub>m</sub>, <sub>app</sub>) and maximal reaction velocity (V<sub>max</sub>), were determined to be 21.59 ± 7.34 μM and 0.031 ± 0.002 μM s<sup>−1</sup> mg<sup>−1</sup>, 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106843"},"PeriodicalIF":1.2,"publicationDate":"2025-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145615543","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

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。

{"title":"Synergistic effect of LTB and Mn2+ adjuvants on immunogenicity of multi-epitope peptides from Staphylococcus aureus","authors":"Jinzhu Ma , Shuyu Wei , Ming Xu , Changquan Liu , Mingyang Tao , Youxi Fu , Kexin Liu , Siqi Wang , Simiao Yu , Liquan Yu , Beiyan Wang , Baifen Song","doi":"10.1016/j.pep.2025.106862","DOIUrl":"10.1016/j.pep.2025.106862","url":null,"abstract":"<div><div>Here, a novel combined adjuvant of heat-labile enterotoxin B (LTB) as an intramolecular adjuvant plus Mn<sup>2+</sup> was prepared to enhance the immunogenicity of multi-epitope (MEP) peptides derived from <em>Staphylococcus aureus</em> (<em>S. aureus</em>). Initially, the <em>mep</em> and <em>ltb-mep</em> fragments were amplified and inserted into pET-28a vectors, respectively, to generate the recombinant plasmids pET28a-<em>mep</em> and pET28a-<em>ltb-mep</em>. 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 Mn<sup>2+</sup> adjuvants. After the second immunization, the mice immunized with LTB-MEP plus Mn<sup>2+</sup> 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 <em>S. aureus</em> challenge. The data showed that the novel LTB-Mn<sup>2+</sup> combined adjuvant exerted a synergistic effect, significantly enhanced the immunogenicity of MEP, and provided a new strategy for improving the efficacy of vaccines against <em>S. aureus</em>.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106862"},"PeriodicalIF":1.2,"publicationDate":"2025-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145615582","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 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。

{"title":"Expression, purification, and biophysical characterization of liquid-liquid phase separation of full-length hnRNPA2B1","authors":"Luis Fernando Durán-Armenta , Attila Meszaros , Julia Malo Pueyo , Steven Janvier , Tamas Lazar , Dominique Maes , Remy Loris , Peter Tompa","doi":"10.1016/j.pep.2025.106861","DOIUrl":"10.1016/j.pep.2025.106861","url":null,"abstract":"<div><div>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 <em>in vitro</em> 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106861"},"PeriodicalIF":1.2,"publicationDate":"2025-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145615545","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

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。

{"title":"Purification of antifreeze proteins from tussah silkworm (Antheraea pernyi) via polyethylene glycol precipitation coupled with aqueous two-phase extraction","authors":"Xinhui Liu, Yiqun Yin, Xin Xu, Tingting Tan, Taoyu Zhou, Yan Wang","doi":"10.1016/j.pep.2025.106856","DOIUrl":"10.1016/j.pep.2025.106856","url":null,"abstract":"<div><div>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 (<em>Antheraea pernyi</em>) 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106856"},"PeriodicalIF":1.2,"publicationDate":"2025-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145588435","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

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。

{"title":"Bench-top NMR of water signals: A non-destructive tool for biomacromolecule characterization","authors":"Tomoto Ura , Taiji Oyama , Chiaki Nishimura","doi":"10.1016/j.pep.2025.106855","DOIUrl":"10.1016/j.pep.2025.106855","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106855"},"PeriodicalIF":1.2,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145565166","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

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。

{"title":"The advantage of high pH eluting Protein A resins over their regular counterparts in aggregate and host cell protein clearance","authors":"Ziyang Li , Lixia Hu , Yifeng Li","doi":"10.1016/j.pep.2025.106859","DOIUrl":"10.1016/j.pep.2025.106859","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106859"},"PeriodicalIF":1.2,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557751","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