Pub Date : 2026-01-13DOI: 10.1016/j.pep.2026.106889
Prashant Jadhav , Hitesh Malviya , Jyoti Gokhale
Streptococcus pneumoniae causes 1.5 million deaths annually, primarily in low-income countries. Pneumococcal histidine triad protein D (PhtD), a conserved 92–110 kDa surface protein, is a promising serotype-independent vaccine candidate due to its roles in adherence, zinc homeostasis, and complement evasion. This study optimized scalable production of His-tagged PhtD in Escherichia coli BL21(DE3). The PhtD gene (serotype 2, strain D39) was cloned into pET30a(+), and expression was optimized using a full factorial design of experiments, achieving up to 19.8 mg/L soluble PhtD (>90 % purity) with 0.1 mM IPTG at OD590 = 0.2 for 2 h. Lysozyme treatment (0.3 mg/mL) increased yield to 22 mg/L, a 120 % improvement over the control (10 mg/L). Purification involved sonication, ultrafiltration, and IMAC (80–100 mM imidazole wash, 200–300 mM elution), with 1 mM PMSF preventing ∼30 % yield loss from proteolysis. PhtD's identity (105–110 kDa), secondary structure (15.5 % helix, 30.4 % sheet), and thermal stability (Tm ∼102 °C) were confirmed by SDS-PAGE, Western blotting, FTIR, circular dichroism, NMR, and DSC. This optimized, statistically guided process delivers the highest reported yield of soluble full-length PhtD in cytoplasmic E. coli with documented native-like folding and exceptional stability (Tm = 102 °C), providing a robust and scalable platform for serotype-independent pneumococcal vaccine development.
{"title":"Scalable production and characterization of recombinant pneumococcal histidine triad protein D in Escherichia coli for vaccine development","authors":"Prashant Jadhav , Hitesh Malviya , Jyoti Gokhale","doi":"10.1016/j.pep.2026.106889","DOIUrl":"10.1016/j.pep.2026.106889","url":null,"abstract":"<div><div><em>Streptococcus pneumoniae</em> causes 1.5 million deaths annually, primarily in low-income countries. Pneumococcal histidine triad protein D (PhtD), a conserved 92–110 kDa surface protein, is a promising serotype-independent vaccine candidate due to its roles in adherence, zinc homeostasis, and complement evasion. This study optimized scalable production of His-tagged PhtD in <em>Escherichia coli</em> BL21(DE3). The PhtD gene (serotype 2, strain D39) was cloned into pET30a(+), and expression was optimized using a full factorial design of experiments, achieving up to 19.8 mg/L soluble PhtD (>90 % purity) with 0.1 mM IPTG at OD<sub>590</sub> = 0.2 for 2 h. Lysozyme treatment (0.3 mg/mL) increased yield to 22 mg/L, a 120 % improvement over the control (10 mg/L). Purification involved sonication, ultrafiltration, and IMAC (80–100 mM imidazole wash, 200–300 mM elution), with 1 mM PMSF preventing ∼30 % yield loss from proteolysis. PhtD's identity (105–110 kDa), secondary structure (15.5 % helix, 30.4 % sheet), and thermal stability (Tm ∼102 °C) were confirmed by SDS-PAGE, Western blotting, FTIR, circular dichroism, NMR, and DSC. This optimized, statistically guided process delivers the highest reported yield of soluble full-length PhtD in cytoplasmic <em>E. coli</em> with documented native-like folding and exceptional stability (Tm = 102 °C), providing a robust and scalable platform for serotype-independent pneumococcal vaccine development.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106889"},"PeriodicalIF":1.2,"publicationDate":"2026-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145981457","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}
Protein-stabilizing additives have traditionally been investigated under dilute solution conditions using simple monomeric proteins as model systems. While such studies have clarified fundamental additive–protein interactions, their direct applicability to practical research and industrial contexts remains limited. This review leverages insights from model systems to summarize additive-based stabilization strategies in two major directions. First, we address process-related stresses, focusing on high-concentration conditions and interfacial stress frequently encountered during protein handling. Second, we highlight strategies for protein classes that are challenging to stabilize or purify, including membrane proteins, viral spike proteins, and intrinsically disordered proteins. This review aims to encourage additive use when proteins are difficult to stabilize under challenging conditions.
{"title":"Additive-based protein stabilization for stress-prone and unstable proteins","authors":"Tomoto Ura, Takumi Nakamura, Moe Iijima, Toya Yoshida, Kentaro Shiraki","doi":"10.1016/j.pep.2026.106885","DOIUrl":"10.1016/j.pep.2026.106885","url":null,"abstract":"<div><div>Protein-stabilizing additives have traditionally been investigated under dilute solution conditions using simple monomeric proteins as model systems. While such studies have clarified fundamental additive–protein interactions, their direct applicability to practical research and industrial contexts remains limited. This review leverages insights from model systems to summarize additive-based stabilization strategies in two major directions. First, we address process-related stresses, focusing on high-concentration conditions and interfacial stress frequently encountered during protein handling. Second, we highlight strategies for protein classes that are challenging to stabilize or purify, including membrane proteins, viral spike proteins, and intrinsically disordered proteins. This review aims to encourage additive use when proteins are difficult to stabilize under challenging conditions.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"240 ","pages":"Article 106885"},"PeriodicalIF":1.2,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145960075","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 : 2026-01-07DOI: 10.1016/j.pep.2026.106878
Mohammed Alosaimi, Waleed Abdulkhair, Jamal Asseri, Abdulmajeed Almuqrin
This study investigated the isolation, enhancement, and optimisation of an α-glucosidase inhibitory protein with hypoglycaemic activity from Streptomyces species obtained from tomato-cultivated soils rich in actinomycetes. A total of 110 Streptomyces 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 Streptomyces griseorubens. Scanning electron microscopy revealed spiral spore chains with smooth, ellipsoidal spores, and 16S rRNA phylogenetic analysis confirmed a close relationship to S. griseorubens (≥90% bootstrap support). UV mutagenesis markedly enhanced the inhibitory activity, with a 20-second 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 sulphate 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-1 and 1,020 U mg-1, respectively. UV mutagenesis significantly improved the α-glucosidase inhibitory activity of S. griseorubens Strep-5, producing a stable, high-molecular-weight protein with strong antidiabetic potential. This work demonstrates the successful isolation, mutagenic enhancement, and optimisation of a microbial α-glucosidase inhibitor from S. griseorubens. The findings highlight its potential as a promising biotechnological and therapeutic agent for diabetes management.
本文研究了从富含放线菌的番茄栽培土壤中获得的链霉菌中具有降糖活性的α-葡萄糖苷酶抑制蛋白的分离、强化和优化。从萨哈市10份土壤样品中共分离出链霉菌110株。16株菌株表现出明显的α-葡萄糖苷酶抑制活性，其中Strep-5的抑制活性最强。形态学、生化、生理和分子分析鉴定Strep-5为灰绿链霉菌。扫描电镜显示螺旋状孢子链，孢子光滑，椭圆形，16S rRNA系统发育分析证实与S. griseorubens亲缘关系密切（≥90% bootstrap支持）。紫外诱变明显增强了抑制活性，暴露20秒产生最活跃的突变体。发酵72小时后，在35°C、pH 7.5和160 rpm的条件下，酵母麦芽提取液的最佳产量出现。采用硫酸铵沉淀法、离子交换法和凝胶过滤色谱法进行顺序纯化，纯化率为8.5倍，回收率为40.8%。纯化后的酶在SDS-PAGE上显示一条70 kDa的条带，证实了其同源性，总活性和比活性分别为21,000 U mL-1和1,020 U mg-1。紫外诱变可显著提高S. griseorubens . Strep-5 α-葡萄糖苷酶抑制活性，生成稳定的高分子量蛋白，具有较强的抗糖尿病潜能。这项工作证明了成功的分离，诱变增强，并优化微生物α-葡萄糖苷酶抑制剂从稻瘟病菌。这些发现突出了它作为一种有前途的生物技术和治疗糖尿病的药物的潜力。
{"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","doi":"10.1016/j.pep.2026.106878","DOIUrl":"https://doi.org/10.1016/j.pep.2026.106878","url":null,"abstract":"<p><p>This study investigated the isolation, enhancement, and optimisation of an α-glucosidase inhibitory protein with hypoglycaemic activity from Streptomyces species obtained from tomato-cultivated soils rich in actinomycetes. A total of 110 Streptomyces 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 Streptomyces griseorubens. Scanning electron microscopy revealed spiral spore chains with smooth, ellipsoidal spores, and 16S rRNA phylogenetic analysis confirmed a close relationship to S. griseorubens (≥90% bootstrap support). UV mutagenesis markedly enhanced the inhibitory activity, with a 20-second 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 sulphate 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 1,020 U mg<sup>-1</sup>, respectively. UV mutagenesis significantly improved the α-glucosidase inhibitory activity of S. griseorubens Strep-5, producing a stable, high-molecular-weight protein with strong antidiabetic potential. This work demonstrates the successful isolation, mutagenic enhancement, and optimisation of a microbial α-glucosidase inhibitor from S. griseorubens. The findings highlight its potential as a promising biotechnological and therapeutic agent for diabetes management.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106878"},"PeriodicalIF":1.2,"publicationDate":"2026-01-07","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}
Pub Date : 2026-01-01DOI: 10.1016/j.pep.2025.106877
Romina Golafshan , Mahmoudreza Aghamali , Mohammad Javad Rasaee
The worldwide COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). The receptor binding domain (RBD) of SARS-Cov-2 is located on the spike protein, which binds and interacts with ACE2. The RBD serve as a crucial target for the development of virus-neutralizing antibodies, vaccinations, and inhibiting agents. The goal of this study was to synthesis recombinant RBD (rRBD) protein and evaluate its expression, function, and stability in different strains of Escherichia coli (E. coli). The rRBD domain gene sequence was engineered utilizing in silico methodologies. The optimized sequence was inserted into pET-28a vector using XhoI and EcoRI restriction sites. Three E. coli expression strains BL21 (DE3), Rosetta-gami, and Shuffle T7 were chosen for protein synthesis. Cloning of the target gene fragment was confirmed using PCR. The validated recombinant vector was cloned into three E. coli strains. SDS-PAGE and Western blot analysis were used to examine the expression of the rRBD. Far-UV circular dichroism (CD) spectroscopy was used to examine secondary structure. ELISA experiments quantified the interaction of rRBD protein with HRP-conjugated anti-His antibody and serum from COVID-19 patients detected with anti-human immunoglobulin-HRP labeled. Protein stability was assessed under different temperature conditions. The rRBD protein was best expressed in all three bacterial strains at 1 mM concentration of IPTG. A 14 kDa band corresponding to the rRBD protein was identified in all three bacterial strains via Western blot. BL21 had the greatest amount of rRBD expression. The rRBD generated from Rosetta-gami exhibited the most robust affinity for anti-His antibodies. All three expressed proteins exhibited similar secondary structure, characterized by a predominance of random coil and β-sheet content. They also exhibited similar affinity to antibodies against SARS-Cov-2 generated from patients. Storage at −20 °C and 4 °C with suitable glycerol concentrations ensured optimal long-term protein stability. This study reveals that E. coli is still a good and flexible host system to produce functional SARS-Cov-2 RBD protein. Even though the strains had different levels of expression and binding efficacy, all of the proteins they made had comparable structures and antigenic properties. The findings validate the feasibility of using bacterial expression systems for the cost-effective production of RBD-based diagnostic reagents.
{"title":"Expression and production of recombinant SARS-CoV-2 RBD protein in E. coli system: comparison of performance, functionality and stability in three strains","authors":"Romina Golafshan , Mahmoudreza Aghamali , Mohammad Javad Rasaee","doi":"10.1016/j.pep.2025.106877","DOIUrl":"10.1016/j.pep.2025.106877","url":null,"abstract":"<div><div>The worldwide COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). The receptor binding domain (RBD) of SARS-Cov-2 is located on the spike protein, which binds and interacts with ACE2. The RBD serve as a crucial target for the development of virus-neutralizing antibodies, vaccinations, and inhibiting agents. The goal of this study was to synthesis recombinant RBD (rRBD) protein and evaluate its expression, function, and stability in different strains of <em>Escherichia coli</em> (<em>E. coli</em>). The rRBD domain gene sequence was engineered utilizing in silico methodologies. The optimized sequence was inserted into pET-28a vector using <em>Xho</em>I and <em>Eco</em>RI restriction sites. Three <em>E. coli</em> expression strains BL21 (DE3), Rosetta-gami, and Shuffle T7 were chosen for protein synthesis. Cloning of the target gene fragment was confirmed using PCR. The validated recombinant vector was cloned into three <em>E. coli</em> strains. SDS-PAGE and Western blot analysis were used to examine the expression of the rRBD. Far-UV circular dichroism (CD) spectroscopy was used to examine secondary structure. ELISA experiments quantified the interaction of rRBD protein with HRP-conjugated anti-His antibody and serum from COVID-19 patients detected with anti-human immunoglobulin-HRP labeled. Protein stability was assessed under different temperature conditions. The rRBD protein was best expressed in all three bacterial strains at 1 mM concentration of IPTG. A 14 kDa band corresponding to the rRBD protein was identified in all three bacterial strains via Western blot. BL21 had the greatest amount of rRBD expression. The rRBD generated from Rosetta-gami exhibited the most robust affinity for anti-His antibodies. All three expressed proteins exhibited similar secondary structure, characterized by a predominance of random coil and β-sheet content. They also exhibited similar affinity to antibodies against SARS-Cov-2 generated from patients. Storage at −20 °C and 4 °C with suitable glycerol concentrations ensured optimal long-term protein stability. This study reveals that <em>E. coli</em> is still a good and flexible host system to produce functional SARS-Cov-2 RBD protein. Even though the strains had different levels of expression and binding efficacy, all of the proteins they made had comparable structures and antigenic properties. The findings validate the feasibility of using bacterial expression systems for the cost-effective production of RBD-based diagnostic reagents.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106877"},"PeriodicalIF":1.2,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145896848","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 : 2025-12-28DOI: 10.1016/j.pep.2025.106876
Jong Shin, David Sanford, Alex A Bohm, William Bachovchin, Peter A Bullock
Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have dramatically improved approaches to cancer therapy (reviewed in Tan et al. (2016), Topalian (2015), Pardoll, (2012) Sharma and Allioson (2015); references (1-4)). One such antibody is termed Nivolumab (trade name Opdivo) that selectively targets PD-1. We previously described a Nivolumab-based anti-PD-1 single chain variable fragment (scFv), termed the anti-PD-1 double mutant (dm) scFv, that tightly binds to PD-1 and blocks the interaction between PD-1 expressed on T-cells and PD-L1 on CHO cells. Extending these experiments, we now report a fluorescent reporter formed by the fusion of the anti-PD-1dm with superfolder (sf) GFP. Immunofluorescence and flow cytometry studies established that the anti-PD-1dm-sfGFP fusion protein can be used to detect PD-1 on the surface of PD-1 expressing Jurkat cells. Furthermore, using a humanized mouse strain that expresses human PD-1 and PD-L1, we report that when combined with an anti-CD3 antibody, the anti-PD-1dm-sfGFP can be used to identify PD-1 expressing T-cells within mouse tumor biopsies. Thus, we have developed a reagent for the quantitative determination of activated T-cells in tumor biopsies. Finally, we report that an additional fluorescent probe for the detection of PD-1 in vivo can be generated by linking, via maleimide chemistry, a derivative of the anti-PD-1dm to the near-infrared dye IR800.
{"title":"DETECTION OF HUMAN PD-1 ON T-CELLS USING A FUSION PROTEIN CONSISTING OF AN ANTI-PD-1 SINGLE CHAIN VARIABLE FRAGMENT LINKED TO SUPERFOLDER GFP.","authors":"Jong Shin, David Sanford, Alex A Bohm, William Bachovchin, Peter A Bullock","doi":"10.1016/j.pep.2025.106876","DOIUrl":"https://doi.org/10.1016/j.pep.2025.106876","url":null,"abstract":"<p><p>Monoclonal antibodies that disrupt the interaction between T-cell derived PD-1 and immunosuppressive ligands, such as PD-L1, have dramatically improved approaches to cancer therapy (reviewed in Tan et al. (2016), Topalian (2015), Pardoll, (2012) Sharma and Allioson (2015); references (1-4)). One such antibody is termed Nivolumab (trade name Opdivo) that selectively targets PD-1. We previously described a Nivolumab-based anti-PD-1 single chain variable fragment (scFv), termed the anti-PD-1 double mutant (dm) scFv, that tightly binds to PD-1 and blocks the interaction between PD-1 expressed on T-cells and PD-L1 on CHO cells. Extending these experiments, we now report a fluorescent reporter formed by the fusion of the anti-PD-1dm with superfolder (sf) GFP. Immunofluorescence and flow cytometry studies established that the anti-PD-1dm-sfGFP fusion protein can be used to detect PD-1 on the surface of PD-1 expressing Jurkat cells. Furthermore, using a humanized mouse strain that expresses human PD-1 and PD-L1, we report that when combined with an anti-CD3 antibody, the anti-PD-1dm-sfGFP can be used to identify PD-1 expressing T-cells within mouse tumor biopsies. Thus, we have developed a reagent for the quantitative determination of activated T-cells in tumor biopsies. Finally, we report that an additional fluorescent probe for the detection of PD-1 in vivo can be generated by linking, via maleimide chemistry, a derivative of the anti-PD-1dm to the near-infrared dye IR800.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106876"},"PeriodicalIF":1.2,"publicationDate":"2025-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145865191","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 : 2025-12-26DOI: 10.1016/j.pep.2025.106875
Odedara Mitalben Bhikhabhai, Hemant Kumar
Viruses of the flavivirus family significantly affect human health worldwide. Dengue virus (DENV), a member of the flavivirus family, causes pathogenic symptoms like high fever, severe headache and body pain upon infection. In some cases, it develops into severe dengue that is fatal. As a crucial pillar of immune response antibodies are produced against different epitopes of the dengue proteins and a collective response is generated by targeting critical proteins. A vaccine against dengue is still to be developed and new antibodies are the need of the hour. In our work we have isolated novel full length heavy chain variable region genes from peripheral blood mononuclear cell (PMBC) of NS1 positive DENV infected patients. The sequencing suggests that the isolated heavy chains to be novel and not yet reported. The sequence verified clones were sub cloned into expression vector pET-28a(+) for protein expression in E. coli. Selected heavy chains were overexpressed and purified to near homogeneity and further specific binding of purified heavy chain variable region with rNS1 was confirmed through competitive inhibition ELISA, dose dependent competitive inhibition ELISA and western blotting. ITC further confirmed the binding of rNS1 and SdAb3 with favourable free energy (ΔG = −9.22 kcal/mol). Identity of purified protein was confirmed by western blotting through anti-His antibody. It was found that a large amount of the purified protein was going in insoluble fraction and a protein refolding strategy was optimized with different buffer conditions. A buffer containing arginine, histidine and sucrose was found to be most efficient in solubilization of protein from insoluble fraction.
{"title":"Novel anti-NS1 human single domain antibody from dengue infected NS1 positive patients","authors":"Odedara Mitalben Bhikhabhai, Hemant Kumar","doi":"10.1016/j.pep.2025.106875","DOIUrl":"10.1016/j.pep.2025.106875","url":null,"abstract":"<div><div>Viruses of the flavivirus family significantly affect human health worldwide. Dengue virus (DENV), a member of the flavivirus family, causes pathogenic symptoms like high fever, severe headache and body pain upon infection. In some cases, it develops into severe dengue that is fatal. As a crucial pillar of immune response antibodies are produced against different epitopes of the dengue proteins and a collective response is generated by targeting critical proteins. A vaccine against dengue is still to be developed and new antibodies are the need of the hour. In our work we have isolated novel full length heavy chain variable region genes from peripheral blood mononuclear cell (PMBC) of NS1 positive DENV infected patients. The sequencing suggests that the isolated heavy chains to be novel and not yet reported. The sequence verified clones were sub cloned into expression vector pET-28a(+) for protein expression in <em>E. coli</em>. Selected heavy chains were overexpressed and purified to near homogeneity and further specific binding of purified heavy chain variable region with rNS1 was confirmed through competitive inhibition ELISA, dose dependent competitive inhibition ELISA and western blotting. ITC further confirmed the binding of rNS1 and SdAb3 with favourable free energy (ΔG = −9.22 kcal/mol). Identity of purified protein was confirmed by western blotting through anti-His antibody. It was found that a large amount of the purified protein was going in insoluble fraction and a protein refolding strategy was optimized with different buffer conditions. A buffer containing arginine, histidine and sucrose was found to be most efficient in solubilization of protein from insoluble fraction.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106875"},"PeriodicalIF":1.2,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145850662","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 : 2025-12-22DOI: 10.1016/j.pep.2025.106872
Chi Linh Thi Nguyen , Uyen Quynh Nguyen , Phuong Mai Vu , Truong Huu Nguyen , Phuong Hoang Nguyen , Chi Quynh Thi Do , Vinh Van Hoang
Blomia tropicalis is a predominant house dust mite in tropical-subtropical climates, and its allergen Blo t 1 is recognized as a key trigger of allergic responses. This study aimed to express, purify recombinant Blo t 1 from B. tropicalis collected in Vietnam and assess its IgE-binding capacity using Vietnamese allergic sera. The cDNA encoding Blo t 1 was amplified from local samples, cloned into pET32b (+), and expressed in Escherichia coli BL21 (DE3). IgE-binding properties were evaluated through dot blot and Western blot assays. The recombinant Blo t 1 showed anomalous SDS-PAGE migration at ∼63 kDa, comprising ∼38.11 kDa from 333 amino acids of the pre-enzyme form of Blo t 1 and ∼19.47 kDa derived from vector-associated sequences, including His-tag. The protein was successfully expressed and purified using His-tag affinity chromatography. Dot blot analysis showed that 19/30 sera (63.33 %) from B. tropicalis-sensitized patients reacted with the recombinant protein, whereas none of the 24 control sera did. Western blot confirmed IgE-binding specificity. The recombinant Blo t 1 was purified under denaturing conditions, so the native three-dimensional structure is unlikely to be retained, and the observed IgE reactivity mainly reflects linear epitope recognition. Even so, the protein represents a useful research material corresponding to a Blo t 1 variant circulating in Vietnam. Along with expanding the limited molecular and IgE-reactivity data available for B. tropicalis in Southeast Asia, it also provides a basis for studies on allergen structure, epitope features, and early exploratory work related to allergen-specific immunotherapy and SIT-oriented approaches.
{"title":"Expression of recombinant preenzyme Blo t 1 allergen in Escherichia coli and its IgE reactivity","authors":"Chi Linh Thi Nguyen , Uyen Quynh Nguyen , Phuong Mai Vu , Truong Huu Nguyen , Phuong Hoang Nguyen , Chi Quynh Thi Do , Vinh Van Hoang","doi":"10.1016/j.pep.2025.106872","DOIUrl":"10.1016/j.pep.2025.106872","url":null,"abstract":"<div><div><em>Blomia tropicalis</em> is a predominant house dust mite in tropical-subtropical climates, and its allergen Blo t 1 is recognized as a key trigger of allergic responses. This study aimed to express, purify recombinant Blo t 1 from <em>B. tropicalis</em> collected in Vietnam and assess its IgE-binding capacity using Vietnamese allergic sera. The cDNA encoding Blo t 1 was amplified from local samples, cloned into pET32b (+), and expressed in <em>Escherichia coli</em> BL21 (DE3). IgE-binding properties were evaluated through dot blot and Western blot assays. The recombinant Blo t 1 showed anomalous SDS-PAGE migration at ∼63 kDa, comprising ∼38.11 kDa from 333 amino acids of the pre-enzyme form of Blo t 1 and ∼19.47 kDa derived from vector-associated sequences, including His-tag. The protein was successfully expressed and purified using His-tag affinity chromatography. Dot blot analysis showed that 19/30 sera (63.33 %) from <em>B. tropicalis</em>-sensitized patients reacted with the recombinant protein, whereas none of the 24 control sera did. Western blot confirmed IgE-binding specificity. The recombinant Blo t 1 was purified under denaturing conditions, so the native three-dimensional structure is unlikely to be retained, and the observed IgE reactivity mainly reflects linear epitope recognition. Even so, the protein represents a useful research material corresponding to a Blo t 1 variant circulating in Vietnam. Along with expanding the limited molecular and IgE-reactivity data available for <em>B. tropicalis</em> in Southeast Asia, it also provides a basis for studies on allergen structure, epitope features, and early exploratory work related to allergen-specific immunotherapy and SIT-oriented approaches.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106872"},"PeriodicalIF":1.2,"publicationDate":"2025-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145828358","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 : 2025-12-19DOI: 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":"10.1016/j.pep.2025.106873","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 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}
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