Protein expression and purification最新文献_第2页

Purification and characterization of the peroxiredoxin 5-thioredoxin 2 complex: unraveling the redox regeneration of peroxiredoxin 5 by thioredoxin 2 within the peroxiredoxin catalytic cycle 过氧还蛋白5-硫氧还蛋白2复合物的纯化与表征：揭示过氧还蛋白催化循环中硫氧还蛋白2对过氧还蛋白5的氧化还原再生

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-28 DOI: 10.1016/j.pep.2026.106896

Xiangwei Yang , Yifan Yang , Sizhe Gong , Li Wang , Qian Liu , Ruiqi Sun , Fu-Ming Lian

{"title":"Purification and characterization of the peroxiredoxin 5-thioredoxin 2 complex: unraveling the redox regeneration of peroxiredoxin 5 by thioredoxin 2 within the peroxiredoxin catalytic cycle","authors":"Xiangwei Yang , Yifan Yang , Sizhe Gong , Li Wang , Qian Liu , Ruiqi Sun , Fu-Ming Lian","doi":"10.1016/j.pep.2026.106896","DOIUrl":"10.1016/j.pep.2026.106896","url":null,"abstract":"<div><div>Peroxiredoxin (Prx) is a critical cysteine-based peroxidase that detoxifies reactive oxygen species to maintain cellular peroxide homeostasis and prevent oxidative stress. Peroxiredoxin 5 (Prx5 or PRDX5) is the unique atypical 2-cysteine Prx in humans, which is able to scavenge the peroxides through redox-active cysteines. However, the precise regeneration mechanism of Prx5 by thioredoxin (Trx) within the catalytic cycle of Prx is not yet well understood. Here, we developed a design strategy through disulfide bond crosslinking to demonstrate that both Cys151 and Cys47 of Prx5 are able to form intermolecular disulfide bonds with thioredoxin 2 (Trx2). Gel filtration was further performed to validate the formation of Prx5-Trx2 complex in solution. We presented a scheme for obtaining the purified Prx5-Trx2 complex, and elucidated that this complex is a heterotetramer with a 1:1 molar ratio of both proteins. Predicted structure model of Prx5-Trx2 complex revealed that each Trx2 protein engages exclusively with a single subunit of the Prx5 dimer, which is distinctly different from the interaction mode observed in yeast Ahp1-Trx2 complex. Collectively, these results provide novel mechanistic insights into the transient Prx5-Trx2 complex formation and the electron transfer from electron donor Trx2 to Prx5 during the catalytic redox regeneration step.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106896"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146079641","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

Additive-based protein stabilization for stress-prone and unstable proteins 基于添加剂的蛋白质稳定应力易感性和不稳定蛋白。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-10 DOI: 10.1016/j.pep.2026.106885

Tomoto Ura, Takumi Nakamura, Moe Iijima, Toya Yoshida, Kentaro Shiraki

引用次数: 0

Recombinant expression and purification of TANB77-derived pilin-like proteins 935 and 938 tanb77衍生的蛋白935和938的重组表达和纯化。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-02-01 DOI: 10.1016/j.pep.2026.106898

Tham Truong Phuong Tran, Linh Thanh Nguyen, Byeong-Gu Han, Min-Jung Lee, Byoung Choul Kim

{"title":"Recombinant expression and purification of TANB77-derived pilin-like proteins 935 and 938","authors":"Tham Truong Phuong Tran, Linh Thanh Nguyen, Byeong-Gu Han, Min-Jung Lee, Byoung Choul Kim","doi":"10.1016/j.pep.2026.106898","DOIUrl":"10.1016/j.pep.2026.106898","url":null,"abstract":"<div><div>Increasing evidence highlights the gut microbiota as a source of diagnostic biomarkers and a key modulator of anti-tumor immunity and cancer immunotherapy responses. Pilin-like proteins from the uncultured TANB77 clade have shown potential to enhance immune checkpoint blockade therapy, yet protocols for their recombinant production have not been established. Here, we present the first optimized method for the expression and purification of two TANB77-derived pilin-like proteins, 935 and 938, using the <em>Escherichia coli</em> (<em>E. coli</em>) BL21(DE3)/pET system. The workflow integrates optimized expression conditions, two-step purification by Ni-NTA affinity and size-exclusion chromatography (SEC), and endotoxin removal for downstream applications. We reveal significant differences in the expression profiles and biophysical properties of the two proteins: protein 935 was expressed at high levels (∼2.5 mg/100 mL) and formed stable oligomers. In contrast, protein 938 exhibited >30-fold lower yields (∼0.08 mg/100 mL) and significant fragmentation, indicating intrinsic instability within this expression system. This study provides the first systematic framework for producing TANB77 pilin-like proteins, facilitating future exploration of their biochemical properties and therapeutic potential in cancer immunotherapy.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106898"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146113908","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

Hyper-expression of functional human basic fibroblast growth factor 2 in Escherichia coli 功能性人碱性成纤维细胞生长因子2在大肠杆菌中的高表达。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-28 DOI: 10.1016/j.pep.2026.106894

Tamilvendan Manavalan , Jiawu Bi , Khongsay Naulchan , Nguyen Thi Bich Van , Xin Yi Chua , Ryan Yow , Sook Yee Lee , Lamony Jian Ming Chew , Christoph Ottenheim , Shigeki Sugii , Manikandan Lakshmanan , Weibiao Zhou , Prakash Arumugam , Fong Tian Wong , Melanie Weingarten , Ee Lui Ang

{"title":"Hyper-expression of functional human basic fibroblast growth factor 2 in Escherichia coli","authors":"Tamilvendan Manavalan , Jiawu Bi , Khongsay Naulchan , Nguyen Thi Bich Van , Xin Yi Chua , Ryan Yow , Sook Yee Lee , Lamony Jian Ming Chew , Christoph Ottenheim , Shigeki Sugii , Manikandan Lakshmanan , Weibiao Zhou , Prakash Arumugam , Fong Tian Wong , Melanie Weingarten , Ee Lui Ang","doi":"10.1016/j.pep.2026.106894","DOIUrl":"10.1016/j.pep.2026.106894","url":null,"abstract":"<div><div>The human basic fibroblast growth factor 2 (hbFGF2) is a highly conserved protein that is crucial for cell proliferation, differentiation, and migration. Commercial hbFGF2 is expensive and contributes significantly to the cost of cultivated meat (CM) media, affecting the scalability of the production process. This study aims to reduce media costs by achieving a high yield of purified active hbFGF2 from <em>Escherichia coli</em> T7 Express. A codon-optimized hbFGF2 variant, hbFGF2-G3, was cloned into the pET-28a (+) plasmid and expressed in <em>E. coli</em> T7 Express. Cultivation parameters, including temperature and induction time, were optimized in both shake flasks and batch bioreactors. Optimized conditions yielded a titer of 1.3 g/L in shake flasks at 20 °C and an impressive 2.95 g/L in a batch bioreactor, quantified by densitometry using an in-house purified hbFGF2-G3 as the standard. The protein was subsequently purified using His-trap affinity and size exclusion chromatography. The functional activity of the purified hbFGF2-G3 was validated through FGF/FGFR activation assays in HepG2 cell lines and cell proliferation studies in Ph9F-1x fish cell lines. Our findings demonstrate a cost-effective method for producing active hbFGF2-G3, which has significant implications for the economic viability of CM production.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106894"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146094030","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

Development and optimization of the hypoglycemic activity of an α-glucosidase inhibitory protein from a UV-induced mutant strain of Streptomyces griseorubens 紫外光诱导的灰黄链霉菌突变株α-葡萄糖苷酶抑制蛋白降糖活性的研究。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-07 DOI: 10.1016/j.pep.2026.106878

Mohammed Alosaimi , Waleed Abdulkhair , Jamal Asseri , Abdulmajeed Almuqrin , Fatemah AlMalki

{"title":"Development and optimization of the hypoglycemic activity of an α-glucosidase inhibitory protein from a UV-induced mutant strain of Streptomyces griseorubens","authors":"Mohammed Alosaimi , Waleed Abdulkhair , Jamal Asseri , Abdulmajeed Almuqrin , Fatemah AlMalki","doi":"10.1016/j.pep.2026.106878","DOIUrl":"10.1016/j.pep.2026.106878","url":null,"abstract":"<div><div>This study investigated the isolation, enhancement, and optimization of an α-glucosidase inhibitory protein with hypoglycaemic activity from <em>Streptomyces</em> species obtained from tomato-cultivated soils rich in actinomycetes. A total of 110 <em>Streptomyces</em> isolates were recovered from ten soil samples collected in Sakha City. Sixteen isolates exhibited notable α-glucosidase inhibitory activity, with isolate Strep-5 showing the highest potency. Morphological, biochemical, physiological, and molecular analyses identified Strep-5 as <em>Streptomyces griseorubens</em>. Scanning electron microscopy revealed spiral spore chains with smooth, ellipsoidal spores, and 16S rRNA phylogenetic analysis confirmed a close relationship to <em>S. griseorubens</em> (≥90 % bootstrap support). UV mutagenesis markedly enhanced the inhibitory activity, with a 20-s exposure producing the most active mutant. Optimal production occurred in yeast–malt extract broth at 35 °C, pH 7.5, and 160 rpm after 72 h of incubation. Sequential purification using ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography yielded an 8.5-fold purification and 40.8 % recovery. The purified enzyme displayed a single 70 kDa band on SDS–PAGE, confirming homogeneity, and exhibited total and specific activities of 21,000 U mL<sup>−1</sup> and 1020 U mg<sup>−1</sup>, respectively. UV mutagenesis significantly improved the α-glucosidase inhibitory activity of <em>S. griseorubens</em> Strep-5, producing a stable, high-molecular-weight protein with strong antidiabetic potential. This work demonstrates the successful isolation, mutagenic enhancement, and optimization of a microbial α-glucosidase inhibitor from <em>S. griseorubens</em>. The findings highlight its potential as a promising biotechnological and therapeutic agent for diabetes management.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106878"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145945919","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 reconstitution of small-conductance mechanosensitive channel into lipid bilayer: ready for solid-state NMR study 小电导机械敏感通道脂质双分子层的表达、纯化和重构：准备用于固态核磁共振研究。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-02-01 DOI: 10.1016/j.pep.2026.106897

Shenghua Meng , Zixiang Gao , Yuan Lin , Yan Zhang , Huayong Xie , Jun Yang , Qiong Tong

{"title":"Expression, purification, and reconstitution of small-conductance mechanosensitive channel into lipid bilayer: ready for solid-state NMR study","authors":"Shenghua Meng , Zixiang Gao , Yuan Lin , Yan Zhang , Huayong Xie , Jun Yang , Qiong Tong","doi":"10.1016/j.pep.2026.106897","DOIUrl":"10.1016/j.pep.2026.106897","url":null,"abstract":"<div><div>Solid-state NMR (ssNMR) provides unique advantages for resolving the structure and interactions of membrane proteins within native-mimicking lipid membranes. However, the challenge is preparing milligrams of stable, homogeneous proteoliposomes samples to achieve high-resolution data. Mechanosensitive channels of small conductance (MscS) are important members of the bacterial mechanosensitive channel family, functioning as emergency release valves to protect cells from osmotic lysis. In this study, AlphaFold was employed to predict the structures of MscS proteins from four bacterial species, shedding light on further experimental work. To obtain homogeneous MscS protein in high yield, several key parameters were systematically optimized, including the expression host strain, induction temperature, and optical density at induction. A “dual-media” approach was also applied. Pa-MscS demonstrated stable high-level expression, yielding about 30 mg/L using “dual-media” method. Gelfiltration chromatography and chemical crosslinking confirmed that Pa-MscS assembles homogeneously into a homoheptamer. Pa-MscS was successfully reconstituted into proteoliposomes, by optimizing the lipid composition of the liposomes, detergent removal conditions, and the phospholipid-to-protein ratio. Finally, 2D <sup>15</sup>N–<sup>13</sup>Cα (NCA) spectroscopy of the Pa-MscS proteoliposomes revealed well-resolved spectra by ssNMR, enabling high-resolution structural studies. This integrative approach provides a robust framework for obtaining high-yield and homogeneous MscS sample for ssNMR-based functional and mechanistic studies, laying the groundwork for future investigations into MscS and therapeutic development.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106897"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146113911","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

Deciphering the inconsistent performance of Protein A membrane on host cell protein clearance 解读蛋白A膜对宿主细胞蛋白清除的不一致表现。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-05-01 Epub Date: 2026-01-24 DOI: 10.1016/j.pep.2026.106891

Yifan Yang, Xudong Zhang, Yifeng Li

{"title":"Deciphering the inconsistent performance of Protein A membrane on host cell protein clearance","authors":"Yifan Yang, Xudong Zhang, Yifeng Li","doi":"10.1016/j.pep.2026.106891","DOIUrl":"10.1016/j.pep.2026.106891","url":null,"abstract":"<div><div>In downstream processing of antibodies and Fc-fusion proteins, Protein A affinity chromatography plays an important role in host cell protein (HCP) reduction. Recently, Protein A membrane has emerged as a promising alternative to resin-based Protein A columns. While Sartobind Rapid A Protein A membrane has been shown to possess stronger capability for removing product-related impurities than traditional Protein A columns, its capability for removing process-related impurities, particularly HCPs, in comparison to Protein A columns is not clear. Limited and conflicting information has been found in the literature in this regard. In the current study, we compared Sartobind Rapid A membrane with MabSelect SuRe LX resin on HCP removal using culture harvests from ten different projects. In all cases but one, the HCP levels of membrane eluates are lower than or comparable to those of column eluates. We speculated that the high flow rate adopted by Protein A membrane serves as both a favorable and an unfavorable factor for minimizing HCP copurification. On one hand, the short residence time reduces HCP leaching during elution; on the other hand, it hinders HCP-antibody disassociation during wash. Protein A membrane provides better and worse HCP clearance than Protein A column when the favorable factor and the unfavorable factor dominates, respectively. For Protein A membrane chromatography to achieve minimum HCP copurification, low and high flow rates should be applied for wash and other steps, respectively. Our work explained the discrepancy between previous studies and shed novel insights on HCP clearance by Protein A membrane.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106891"},"PeriodicalIF":1.2,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146053382","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

Protein expression and purification Pub Date : 2026-03-01 Epub Date: 2025-11-29 DOI: 10.1016/j.pep.2025.106865

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

{"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":"2026-03-01","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

Protein expression and purification Pub Date : 2026-03-01 Epub Date: 2025-11-29 DOI: 10.1016/j.pep.2025.106863

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

{"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":"2026-03-01","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

Comparative analysis of SUMO and his-tag fusion for soluble expression of the SARS-CoV-2 omicron RBD in E. coli SUMO与his-tag融合在大肠杆菌中可溶性表达SARS-CoV-2组粒RBD的比较分析

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2026-03-01 Epub Date: 2025-12-09 DOI: 10.1016/j.pep.2025.106869

Sadegh Zargan , Saba Mesdaghinia , Hasan Jalili , Bahareh Dabirmanesh , Khosro Khajeh

{"title":"Comparative analysis of SUMO and his-tag fusion for soluble expression of the SARS-CoV-2 omicron RBD in E. coli","authors":"Sadegh Zargan , Saba Mesdaghinia , Hasan Jalili , Bahareh Dabirmanesh , Khosro Khajeh","doi":"10.1016/j.pep.2025.106869","DOIUrl":"10.1016/j.pep.2025.106869","url":null,"abstract":"<div><div>The soluble expression of the SARS-CoV-2 Receptor-Binding Domain (RBD) is fundamental for manufacturing protein-based countermeasures. Although <em>E. coli</em> is a favored host for rapid production, its utility is often constrained by the formation of insoluble aggregates, thereby limiting the supply for vaccine and diagnostic development. Given the persistent threat of emerging SARS-CoV-2 variants, the need for reliable, high-yield expression platforms to overcome this solubility challenge remains critical. This study aimed to achieve soluble expression of the Omicron RBD variant in <em>E. coli</em> using a computationally guided strategy. To support solubility-enhancing tag selection, a machine learning–based tool (TagSolver), trained on the UESolDS dataset, predicted a 61.98 % solubility probability for the SUMO–RBD. Two constructs were designed: one containing a SUMO tag and a His-tagged RBD as a control. Both were expressed in <em>E. coli</em> SHuffle® T7 cells, and solubility was assessed by SDS–PAGE after 8 h of induction at 22 °C. The SUMO–RBD was expressed in a soluble form, whereas the His-tagged RBD was insoluble. Following purification and SUMO protease cleavage, FTIR analysis indicated a native-like secondary structure enriched in β-sheets. These findings demonstrate, for the first time, the soluble expression of the SARS-CoV-2 Omicron RBD in <em>E. coli</em> and establish a refolding-free strategy for producing this antigen. Furthermore, the TagSolver predictor was introduced, which may accelerate the selection of solubility-enhancing tags.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106869"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145736792","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