Pub Date : 2025-12-19DOI: 10.1016/j.pep.2025.106874
Qi Xu , Xinying He , Jiale Bai , Hanlin Liu , Tao Hong , Suying Dang , Wei Zhang
Objective
To develop an optimized prokaryotic expression system for producing functional soluble CD40 ligand (sCD40L) and characterize its hemostatic activity.
Methods
The codon-optimized sCD40L gene (encoding residues 113–261) was synthesized and cloned into pET28a vector via BamHI/XhoI sites. After transformation into E. coli BL21(DE3), soluble expression was induced with 0.5 mM IPTG at 20 °C for 12 h. The recombinant protein was purified using nickel-affinity chromatography and analyzed by SDS-PAGE and Western blot. Biological activity was assessed through in vitro platelet aggregation and in vivo hemostasis assays.
Results
The pET28a-sCD40L expression vector was successfully constructed, and the protein purity reached more than 90 %. Western blot confirmed the expected ∼20 kDa sCD40L. Functionally, the recombinant sCD40L enhanced platelet aggregation in a dose-dependent manner, and significantly reduced secondary bleeding time in mice.
Conclusion
Our optimized prokaryotic system efficiently produces functional sCD40L without requiring eukaryotic post-translational modifications, providing a valuable tool for studying CD40L-mediated hemostasis and developing potential therapeutic applications.
{"title":"Expression and functional characterization of recombinant soluble CD40 Ligand in prokaryotic systems","authors":"Qi Xu , Xinying He , Jiale Bai , Hanlin Liu , Tao Hong , Suying Dang , Wei Zhang","doi":"10.1016/j.pep.2025.106874","DOIUrl":"10.1016/j.pep.2025.106874","url":null,"abstract":"<div><h3>Objective</h3><div>To develop an optimized prokaryotic expression system for producing functional soluble CD40 ligand (sCD40L) and characterize its hemostatic activity.</div></div><div><h3>Methods</h3><div>The codon-optimized <em>sCD40L</em> gene (encoding residues 113–261) was synthesized and cloned into pET28a vector via BamHI/XhoI sites. After transformation into <em>E. coli</em> BL21(DE3), soluble expression was induced with 0.5 mM IPTG at 20 °C for 12 h. The recombinant protein was purified using nickel-affinity chromatography and analyzed by SDS-PAGE and Western blot. Biological activity was assessed through in vitro platelet aggregation and in vivo hemostasis assays.</div></div><div><h3>Results</h3><div>The pET28a-sCD40L expression vector was successfully constructed, and the protein purity reached more than 90 %. Western blot confirmed the expected ∼20 kDa sCD40L. Functionally, the recombinant sCD40L enhanced platelet aggregation in a dose-dependent manner, and significantly reduced secondary bleeding time in mice.</div></div><div><h3>Conclusion</h3><div>Our optimized prokaryotic system efficiently produces functional sCD40L without requiring eukaryotic post-translational modifications, providing a valuable tool for studying CD40L-mediated hemostasis and developing potential therapeutic applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106874"},"PeriodicalIF":1.2,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786906","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}
Pub Date : 2025-12-17DOI: 10.1016/j.pep.2025.106873
Ruomei Lv, Wenjing Qi, Yifeng Li
Affinity chromatography is widely utilized for initial product capture in antibody purification. Besides Protein A resins, other affinity resins that bind different subdomains of an antibody have also been developed. For example, Protein L resin, KappaSelect, LambdaFabSelect and Praesto 70 CH1 selectively bind the variable region of kappa light chain (LC), constant region of kappa LC, constant region of lambda LC and CH1 domain of the heavy chain (HC), respectively. Affinity media with distinct specificities provide more options for antibody purification. For a given bispecific antibody (bsAb) purification case, depending on the composition difference between the product and byproducts, one affinity resin may outperform the others for byproduct removal. In the current study, we compared four affinity resins, Praesto Jetted A50 (a Protein A resin), Praesto 70 CH1, KappaSelect and MabSelect VL (Cytiva's second-generation Protein L resin), for their effectiveness in removing byproducts associated with a VHH-containing trispecific antibody (TsAb). The data suggested that the three non-Protein A resins outperformed the Protein A resin for byproduct removal. Among the three non-Protein A resins, MabSelect VL provided the best byproduct clearance, and it removed half-antibody, homodimers and most aggregates. This work reconfirms the necessity of screening different affinity resins with distinct specificities for achieving the best separation.
{"title":"The superiority of MabSelect VL, Cytiva's second-generation Protein L resin, over three other affinity resins in purifying a VHH-containing trispecific antibody.","authors":"Ruomei Lv, Wenjing Qi, Yifeng Li","doi":"10.1016/j.pep.2025.106873","DOIUrl":"https://doi.org/10.1016/j.pep.2025.106873","url":null,"abstract":"<p><p>Affinity chromatography is widely utilized for initial product capture in antibody purification. Besides Protein A resins, other affinity resins that bind different subdomains of an antibody have also been developed. For example, Protein L resin, KappaSelect, LambdaFabSelect and Praesto 70 CH1 selectively bind the variable region of kappa light chain (LC), constant region of kappa LC, constant region of lambda LC and CH1 domain of the heavy chain (HC), respectively. Affinity media with distinct specificities provide more options for antibody purification. For a given bispecific antibody (bsAb) purification case, depending on the composition difference between the product and byproducts, one affinity resin may outperform the others for byproduct removal. In the current study, we compared four affinity resins, Praesto Jetted A50 (a Protein A resin), Praesto 70 CH1, KappaSelect and MabSelect VL (Cytiva's second-generation Protein L resin), for their effectiveness in removing byproducts associated with a VHH-containing trispecific antibody (TsAb). The data suggested that the three non-Protein A resins outperformed the Protein A resin for byproduct removal. Among the three non-Protein A resins, MabSelect VL provided the best byproduct clearance, and it removed half-antibody, homodimers and most aggregates. This work reconfirms the necessity of screening different affinity resins with distinct specificities for achieving the best separation.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106873"},"PeriodicalIF":1.2,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145794505","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}
Pub Date : 2025-12-15DOI: 10.1016/j.pep.2025.106871
Miguel Angel Mejía Sánchez , Samuel Cardoso Arenas , Ricardo Miranda Blancas , Herlinda Clement , Ligia Luz Corrales García , Adrián Marcelo Franco-Vásquez , Ivan Arenas , Alejandro Carbajal Saucedo , Gerardo Corzo
The crotoxin complex is identified as the primary lethal toxin in various species of rattlesnakes. It comprises two subunits: CtxA, a non-toxic protein, and CtxB, a minimally toxic enzymatic phospholipase A2. These components exert neurotoxic effects by disrupting neuromuscular transmission, specifically by inhibiting the release of acetylcholine, thereby causing flaccid paralysis of the muscles. Due to crotoxin's high lethality, variable presence, and proportion within crotalid venoms, considerable interest has been directed toward its inhibition. In this context, the heterologous expression of a recombinant fusion protein designated as rCtxBA, which incorporates the amino acid sequences of both CtxA and CtxB subunits of crotoxin, has been documented. The rCtxBA protein was recombinantly expressed, recovered, and subsequently purified. New Zealand rabbits were immunized with the recombinants rCtxBA, rCTxB, and the native CtxBA. ELISA and Western blot analyses revealed that the anti-crotoxin antibodies specifically recognized venoms containing crotoxin by binding to both conformational and linear epitopes of crotoxin-like or related phospholipases. In vivo neutralization assays performed in mice demonstrated efficacy of the anti-rCtxBA antibodies against venoms from Crotalus mictlantecuhtli, C. scutulatus salvini, and C. tzabcan—species known to contain crotoxin—with ED50 values of 2.8, 7.8, and 2.5 mg/3LD50, respectively. Conversely, no neutralization effect was observed against the venom of C. molossus nigrescens, a species lacking crotoxin in its venom profile. These findings substantiate that native crotoxin, present in whole crotalid venoms, can be neutralized using recombinant crotalid antigens, thereby facilitating the development of effective neutralizing antibodies.
{"title":"Recombinant expression of crotoxin and comparison to native crotoxin as immunogens for anti-crotalid antivenoms","authors":"Miguel Angel Mejía Sánchez , Samuel Cardoso Arenas , Ricardo Miranda Blancas , Herlinda Clement , Ligia Luz Corrales García , Adrián Marcelo Franco-Vásquez , Ivan Arenas , Alejandro Carbajal Saucedo , Gerardo Corzo","doi":"10.1016/j.pep.2025.106871","DOIUrl":"10.1016/j.pep.2025.106871","url":null,"abstract":"<div><div>The crotoxin complex is identified as the primary lethal toxin in various species of rattlesnakes. It comprises two subunits: CtxA, a non-toxic protein, and CtxB, a minimally toxic enzymatic phospholipase A<sub>2</sub>. These components exert neurotoxic effects by disrupting neuromuscular transmission, specifically by inhibiting the release of acetylcholine, thereby causing flaccid paralysis of the muscles. Due to crotoxin's high lethality, variable presence, and proportion within crotalid venoms, considerable interest has been directed toward its inhibition. In this context, the heterologous expression of a recombinant fusion protein designated as rCtxBA, which incorporates the amino acid sequences of both CtxA and CtxB subunits of crotoxin, has been documented. The rCtxBA protein was recombinantly expressed, recovered, and subsequently purified. New Zealand rabbits were immunized with the recombinants rCtxBA, rCTxB, and the native CtxBA. ELISA and Western blot analyses revealed that the anti-crotoxin antibodies specifically recognized venoms containing crotoxin by binding to both conformational and linear epitopes of crotoxin-like or related phospholipases. <em>In vivo</em> neutralization assays performed in mice demonstrated efficacy of the anti-rCtxBA antibodies against venoms from <em>Crotalus mictlantecuhtli, C. scutulatus salvini</em>, and <em>C. tzabcan—</em>species known to contain crotoxin—with ED<sub>50</sub> values of 2.8, 7.8, and 2.5 mg/3LD<sub>50</sub>, respectively. Conversely, no neutralization effect was observed against the venom of <em>C. molossus nigrescens</em>, a species lacking crotoxin in its venom profile. These findings substantiate that native crotoxin, present in whole crotalid venoms, can be neutralized using recombinant crotalid antigens, thereby facilitating the development of effective neutralizing antibodies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106871"},"PeriodicalIF":1.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145775233","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}
Pub Date : 2025-12-09DOI: 10.1016/j.pep.2025.106869
Sadegh Zargan , Saba Mesdaghinia , Hasan Jalili , Bahareh Dabirmanesh , Khosro Khajeh
The soluble expression of the SARS-CoV-2 Receptor-Binding Domain (RBD) is fundamental for manufacturing protein-based countermeasures. Although E. coli 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 E. coli 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 E. coli 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 E. coli 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.
{"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":"2025-12-09","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}
Pub Date : 2025-12-09DOI: 10.1016/j.pep.2025.106870
Nindy Novita Sari , Nurhasanah , Titin Haryati
The discovery of a new microbial lipase is crucial since its utilization has various industrial applications. Bacillus cereus is a strain that has emerged as a potential source of microbial lipase. Until now, the exploration of new lipases from Bacillus cereus remains limited. This study aims to obtain a lipase enzyme from Bacillus cereus ALP E1, as well as to purify and characterize the lipase produced by this strain. In this study, an extracellular acidophilic lipase from Bacillus cereus ALP E1 isolated from seawater at Panjang Port, Lampung, Indonesia was partially purified and characterized. Extracellular lipase was produced using submerged fermentation methods, and then partially purified with ammonium sulphate fractionation and dialysis. Partial purification successfully achieved a 12.31 purification fold with specific activity at 31083.14 U/mg. According to SDS-PAGE analysis, the purified lipase had a single dominant band estimated at 55 kDa. Lipase characterization revealed an optimal pH of 5, an optimal temperature of 40 °C, and the highest activity towards the substrate p-nitrophenyl stearate (C18). The lipase was activated by Ba2+ and deactivated by Mg2+. This lipase activity is enhanced by adding 1 % Tween 20, but it is deactivated by Tween 80. All tested organic solvents at 5 % concentration were able to activate the lipase with the optimized two-fold lipase activation using chloroform added. The results demonstrate that seawater is a promising source for discovering lipases with high activity. This complete characterization is important information and serves as a foundation for using lipase in appropriate industrial applications.
{"title":"Partial purification and characterization acidophilic lipase from Bacillus cereus ALP E1","authors":"Nindy Novita Sari , Nurhasanah , Titin Haryati","doi":"10.1016/j.pep.2025.106870","DOIUrl":"10.1016/j.pep.2025.106870","url":null,"abstract":"<div><div>The discovery of a new microbial lipase is crucial since its utilization has various industrial applications. <em>Bacillus cereus</em> is a strain that has emerged as a potential source of microbial lipase. Until now, the exploration of new lipases from <em>Bacillus cereus</em> remains limited. This study aims to obtain a lipase enzyme from <em>Bacillus cereus</em> ALP E1, as well as to purify and characterize the lipase produced by this strain. In this study, an extracellular acidophilic lipase from <em>Bacillus cereus</em> ALP E1 isolated from seawater at Panjang Port, Lampung, Indonesia was partially purified and characterized. Extracellular lipase was produced using submerged fermentation methods, and then partially purified with ammonium sulphate fractionation and dialysis. Partial purification successfully achieved a 12.31 purification fold with specific activity at 31083.14 U/mg. According to SDS-PAGE analysis, the purified lipase had a single dominant band estimated at 55 kDa. Lipase characterization revealed an optimal pH of 5, an optimal temperature of 40 °C, and the highest activity towards the substrate <em>p</em>-nitrophenyl stearate (C18). The lipase was activated by Ba<sup>2+</sup> and deactivated by Mg<sup>2+</sup>. This lipase activity is enhanced by adding 1 % Tween 20, but it is deactivated by Tween 80. All tested organic solvents at 5 % concentration were able to activate the lipase with the optimized two-fold lipase activation using chloroform added. The results demonstrate that seawater is a promising source for discovering lipases with high activity. This complete characterization is important information and serves as a foundation for using lipase in appropriate industrial applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106870"},"PeriodicalIF":1.2,"publicationDate":"2025-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145725695","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}
Pub Date : 2025-12-08DOI: 10.1016/j.pep.2025.106868
Weijie Zhou, Yuting Wang, Shaobo Liang, Jingjin Hu, Feng Pang
Bovine viral diarrhea virus (BVDV) is an economically important pathogen for causing bovine viral diarrhea (BVD), with a significant detrimental impact on cattle production worldwide. While the BVDV NS5A protein is known to be essential for replication, its function remain incompletely characterized. This study entailed cloning the NS5A gene and subsequently generating the recombinant plasmid pET-28a-NS5A for expression in Escherichia coli. The purified and refolded recombinant NS5A protein was employed to immunize New Zealand white rabbits, resulting in the production of specific polyclonal antibodies. The antibody was then deployed to investigate the expression of the NS5A protein. Our findings confirmed the NS5A protein was successfully produced and purified in a prokaryotic system. The generated anti-NS5A polyclonal antibody in rabbits showed a robust titer of 1:819,200. The antibody specifically detected the NS5A protein expressed in both prokaryotic and eukaryotic systems. With its high specificity and titer, this antibody may serve as a valuable reagent for investigating NS5A's functions in BVDV pathogenesis. It also holds potential for application in developing diagnostic assays and novel control strategies against BVD.
{"title":"Generation of a polyclonal antibody that specifically recognizes the recombinant NS5A protein of bovine viral diarrhea virus","authors":"Weijie Zhou, Yuting Wang, Shaobo Liang, Jingjin Hu, Feng Pang","doi":"10.1016/j.pep.2025.106868","DOIUrl":"10.1016/j.pep.2025.106868","url":null,"abstract":"<div><div>Bovine viral diarrhea virus (BVDV) is an economically important pathogen for causing bovine viral diarrhea (BVD), with a significant detrimental impact on cattle production worldwide. While the BVDV NS5A protein is known to be essential for replication, its function remain incompletely characterized. This study entailed cloning the NS5A gene and subsequently generating the recombinant plasmid pET-28a-NS5A for expression in <em>Escherichia coli</em>. The purified and refolded recombinant NS5A protein was employed to immunize New Zealand white rabbits, resulting in the production of specific polyclonal antibodies. The antibody was then deployed to investigate the expression of the NS5A protein. Our findings confirmed the NS5A protein was successfully produced and purified in a prokaryotic system. The generated anti-NS5A polyclonal antibody in rabbits showed a robust titer of 1:819,200. The antibody specifically detected the NS5A protein expressed in both prokaryotic and eukaryotic systems. With its high specificity and titer, this antibody may serve as a valuable reagent for investigating NS5A's functions in BVDV pathogenesis. It also holds potential for application in developing diagnostic assays and novel control strategies against BVD.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106868"},"PeriodicalIF":1.2,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145725710","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}
Pub Date : 2025-12-04DOI: 10.1016/j.pep.2025.106866
Weichao Zhu , Huahong Yu , Ying Zhang , Qishu Luo , Min Song , Qin Geng , Lihua Zhou , Zhihua Wu
Hazelnut (Corylus avellana) is a widely consumed nut with potent allergenic potential, and the isolation and purification of native hazelnut allergens are critical for the effective prevention and management of hazelnut allergy. In this study, two major allergens Cor a 9 and Cor a 14 were purified from hazelnut kernels via a sequential protocol involving pulverization, defatting, protein extraction, ammonium sulfate precipitation, dialysis desalting, and anion-exchange chromatography. Results demonstrated successful purification of the target allergens with high purity and intact conformational structure, which exhibited specific immunoreactivity with sera from hazelnut-allergic patients. A single preparation run yielded over 5 mg of Cor a 14 (purity >95 %) and more than 1 mg of Cor a 9 (purity >85 %). The established protocol is simple, requires minimal equipment, and offers high efficiency, laying a preliminary foundation for hazelnut allergen-related research. In conclusion, we successfully isolated and purified two major hazelnut allergenic proteins Cor a 9 and Cor a 14, providing a valuable material basis for investigating the sensitization mechanism of hazelnut allergy and developing diagnostic tools or hypoallergenic products.
榛子是一种广泛食用的具有强致敏性的坚果，天然榛子过敏原的分离纯化是有效预防和管理榛子过敏的关键。在本研究中，通过粉碎、脱脂、蛋白质提取、硫酸铵沉淀、透析脱盐和阴离子交换色谱等顺序程序，从榛子仁中纯化了两个主要的过敏原Cor a 9和Cor a 14。结果表明，目标过敏原纯化成功，具有高纯度和完整的构象结构，对榛子过敏患者血清具有特异性免疫反应性。单次制备可产生5mg以上的Cor a14（纯度95%）和1mg以上的Cor a9（纯度85%）。所建立的方案简单、设备少、效率高，为榛子过敏原相关研究奠定了初步基础。综上所述，我们成功分离纯化了两个主要的榛子致敏蛋白Cor a 9和Cor a 14，为研究榛子过敏的致敏机制、开发诊断工具或低致敏产品提供了有价值的物质基础。
{"title":"Isolation, purification and identification of major allergens Cor a 9 and Cor a 14 in hazelnuts","authors":"Weichao Zhu , Huahong Yu , Ying Zhang , Qishu Luo , Min Song , Qin Geng , Lihua Zhou , Zhihua Wu","doi":"10.1016/j.pep.2025.106866","DOIUrl":"10.1016/j.pep.2025.106866","url":null,"abstract":"<div><div>Hazelnut (<em>Corylus avellana</em>) is a widely consumed nut with potent allergenic potential, and the isolation and purification of native hazelnut allergens are critical for the effective prevention and management of hazelnut allergy. In this study, two major allergens Cor a 9 and Cor a 14 were purified from hazelnut kernels via a sequential protocol involving pulverization, defatting, protein extraction, ammonium sulfate precipitation, dialysis desalting, and anion-exchange chromatography. Results demonstrated successful purification of the target allergens with high purity and intact conformational structure, which exhibited specific immunoreactivity with sera from hazelnut-allergic patients. A single preparation run yielded over 5 mg of Cor a 14 (purity >95 %) and more than 1 mg of Cor a 9 (purity >85 %). The established protocol is simple, requires minimal equipment, and offers high efficiency, laying a preliminary foundation for hazelnut allergen-related research. In conclusion, we successfully isolated and purified two major hazelnut allergenic proteins Cor a 9 and Cor a 14, providing a valuable material basis for investigating the sensitization mechanism of hazelnut allergy and developing diagnostic tools or hypoallergenic products.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106866"},"PeriodicalIF":1.2,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145681148","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}
Pub Date : 2025-12-03DOI: 10.1016/j.pep.2025.106867
Sen Zou , Xiaoxiao Li , Jiajun Liu , Shuai Fan , Zhaoyong Yang
Hemophilia, a genetic disorder characterized by impaired blood clotting, necessitates innovative treatments. This study optimized the expression of recombinant human coagulation factor VII (rhFVII) in HEK293 cells to enhance its therapeutic efficacy. The human FVII gene was cloned into the pcDNA3.1 vector, and transient transfection was performed with an optimized DNA-to-PEI ratio (1:2) at 70 % cell density. Stable FVII-expressing cell lines were selected with G418, confirmed via RT-PCR, and adapted to suspension culture. rhFVII expression was analyzed using SDS-PAGE, Western blot, and ELISA, while its coagulation activity was evaluated by prothrombin time tests. Transient transfection yielded rhFVII concentrations of 10.22 μg/L and clotting activity of 232.46 %. Stable monoclonal HEK293 cells in suspension produced rhFVII at 11.22 mg/L with maximum coagulation activity of 563.17 % and high stability and safety. The findings underscore the successful optimization of rhFVII expression in HEK293 cells, paving the way for advancements in biopharmaceutical production and improved treatment options for bleeding disorders. Future research should focus on in vivo validation and further refinement of culture conditions to maximize protein yield and stability.
{"title":"Optimized production and coagulation activity of rhFVII in HEK293 cells for bleeding disorders","authors":"Sen Zou , Xiaoxiao Li , Jiajun Liu , Shuai Fan , Zhaoyong Yang","doi":"10.1016/j.pep.2025.106867","DOIUrl":"10.1016/j.pep.2025.106867","url":null,"abstract":"<div><div>Hemophilia, a genetic disorder characterized by impaired blood clotting, necessitates innovative treatments. This study optimized the expression of recombinant human coagulation factor VII (rhFVII) in HEK293 cells to enhance its therapeutic efficacy. The human FVII gene was cloned into the pcDNA3.1 vector, and transient transfection was performed with an optimized DNA-to-PEI ratio (1:2) at 70 % cell density. Stable FVII-expressing cell lines were selected with G418, confirmed via RT-PCR, and adapted to suspension culture. rhFVII expression was analyzed using SDS-PAGE, Western blot, and ELISA, while its coagulation activity was evaluated by prothrombin time tests. Transient transfection yielded rhFVII concentrations of 10.22 μg/L and clotting activity of 232.46 %. Stable monoclonal HEK293 cells in suspension produced rhFVII at 11.22 mg/L with maximum coagulation activity of 563.17 % and high stability and safety. The findings underscore the successful optimization of rhFVII expression in HEK293 cells, paving the way for advancements in biopharmaceutical production and improved treatment options for bleeding disorders. Future research should focus on in vivo validation and further refinement of culture conditions to maximize protein yield and stability.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106867"},"PeriodicalIF":1.2,"publicationDate":"2025-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145687724","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}
Pub Date : 2025-12-01Epub Date: 2025-08-05DOI: 10.1016/j.pep.2025.106787
Tetsuya Wakabayashi, Taichi Kuramochi
Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.
{"title":"Discovery and development of bispecific antibodies.","authors":"Tetsuya Wakabayashi, Taichi Kuramochi","doi":"10.1016/j.pep.2025.106787","DOIUrl":"10.1016/j.pep.2025.106787","url":null,"abstract":"<p><p>Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106787"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144776016","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}
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.
{"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}
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