Pub Date : 2026-01-01Epub Date: 2025-09-11DOI: 10.1016/j.pep.2025.106812
Kennosuke Ichikawa, Mike J. McGrew
Avian biology has contributed to many research areas, such as sustainable protein production, endocrinology, developmental biology, neurosciences, and immunology. Primordial germ cells, lineage-restricted stem cells, are key for the conservation of genetic diversity of bird species, as well as for studying germ cell development and producing genetic models to study avian biology. Here, we review the current knowledge of developmental and fate decision processes in avian primordial germ cells focusing on insights revealed by gene expression profiling. We summarized the characteristics and fundamental pathways required for chicken primordial cell growth. In addition, we discuss the common and disparate features of PGCs from chicken compared to other avian species. These insights are valuable for researchers in germ cell biology, reproductive biotechnology, and avian genetic conservation and indicate a need for the analysis of further bird species.
{"title":"Transcriptome profiles for defining avian primordial germ cell development","authors":"Kennosuke Ichikawa, Mike J. McGrew","doi":"10.1016/j.pep.2025.106812","DOIUrl":"10.1016/j.pep.2025.106812","url":null,"abstract":"<div><div>Avian biology has contributed to many research areas, such as sustainable protein production, endocrinology, developmental biology, neurosciences, and immunology. Primordial germ cells, lineage-restricted stem cells, are key for the conservation of genetic diversity of bird species, as well as for studying germ cell development and producing genetic models to study avian biology. Here, we review the current knowledge of developmental and fate decision processes in avian primordial germ cells focusing on insights revealed by gene expression profiling. We summarized the characteristics and fundamental pathways required for chicken primordial cell growth. In addition, we discuss the common and disparate features of PGCs from chicken compared to other avian species. These insights are valuable for researchers in germ cell biology, reproductive biotechnology, and avian genetic conservation and indicate a need for the analysis of further bird species.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106812"},"PeriodicalIF":1.2,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058352","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}
Histidinol dehydrogenase (HisD, E.C. 1.1.1.23), encoded by the hisD gene and catalyzing the final two steps in the l-histidine biosynthesis, has emerged as a promising antibacterial target for several human pathogens, such as Brucella suis and Mycobacterium tuberculosis. Herein, biochemical properties on recombinant HisD from the human pathogen Neisseria gonorrhoeae (NgHisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant NgHisD is a homodimer (∼105 kDa native size; ∼47 kDa subunit). Kinetic analysis identified this protein as a highly selective NAD+-dependent enzyme (specific activity: 17.3 ± 0.4 U mg−1), exhibiting minimal activity with NADP+. The KM values for NAD+ and l-histidinol were 0.9 ± 0.3 mM and 45 ± 6 μM, respectively. Maximal activity occurred at pH 9.5 and 45 °C in the presence of Mn2+. Heat-inactivation experiment showed that rapid inactivation began to occur following incubation at 45 °C for 30 min. The metal ions coordinated by the enzyme were not sequestered by 10 mM EDTA but were efficiently chelated by 1,10-phenanthroline. The recombinant NgHisD lost 85 % activity in the presence of 1 mM 1,10-phenantroline. Our study not only establishes a foundation for investigating the physiological function of NgHisD in vivo but also uncovers the absence of the canonical histidine biosynthesis pathway in Neisseria cinerea.
组氨酸脱氢酶（HisD， E.C. 1.1.1.23）由HisD基因编码，催化l-组氨酸生物合成的最后两步，已成为猪布鲁氏菌和结核分枝杆菌等几种人类病原体的有前途的抗菌靶点。本文对人淋病奈瑟菌（Neisseria gonorrhoeae, NgHisD）重组HisD的生化特性进行了详细的表征。SDS-PAGE和大小隔离层析显示重组NgHisD是一个同源二聚体（约105 kDa天然大小；约47 kDa亚基）。动力学分析表明，该蛋白是一种高度选择性的NAD+依赖酶（比活性：17.3±0.4 U mg-1），对NADP+的活性极小。NAD+和l-组氨酸二醇的KM值分别为0.9±0.3 mM和45±6 μM。在pH 9.5和45°C条件下，Mn2+的存在使活性达到最大。热失活实验表明，在45℃孵育30 min后，开始出现快速失活。该酶配位的金属离子不被10 mM EDTA隔离，而被1,10-菲罗啉有效地螯合。重组NgHisD在1mm - 1,10-phenantroline存在下失去85%的活性。我们的研究不仅为研究NgHisD在体内的生理功能奠定了基础，而且揭示了灰奈瑟菌中缺乏典型的组氨酸生物合成途径。
{"title":"Biochemical characterization of histidinol dehydrogenase from the human pathogen Neisseria gonorrhoeae","authors":"Shuping Fei , Jiatong Zhao , Wenwen Wu , Chaoqun Lian , Wanggang Tang","doi":"10.1016/j.pep.2025.106824","DOIUrl":"10.1016/j.pep.2025.106824","url":null,"abstract":"<div><div>Histidinol dehydrogenase (HisD, E.C. 1.1.1.23), encoded by the <em>hisD</em> gene and catalyzing the final two steps in the <span>l</span>-histidine biosynthesis, has emerged as a promising antibacterial target for several human pathogens, such as <em>Brucella suis</em> and <em>Mycobacterium tuberculosis</em>. Herein, biochemical properties on recombinant HisD from the human pathogen <em>Neisseria gonorrhoeae</em> (<em>Ng</em>HisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant <em>Ng</em>HisD is a homodimer (∼105 kDa native size; ∼47 kDa subunit). Kinetic analysis identified this protein as a highly selective NAD<sup>+</sup>-dependent enzyme (specific activity: 17.3 ± 0.4 U mg<sup>−1</sup>), exhibiting minimal activity with NADP<sup>+</sup>. The <em>K</em><sub>M</sub> values for NAD<sup>+</sup> and <span>l</span>-histidinol were 0.9 ± 0.3 mM and 45 ± 6 μM, respectively. Maximal activity occurred at pH 9.5 and 45 °C in the presence of Mn<sup>2+</sup>. Heat-inactivation experiment showed that rapid inactivation began to occur following incubation at 45 °C for 30 min. The metal ions coordinated by the enzyme were not sequestered by 10 mM EDTA but were efficiently chelated by 1,10-phenanthroline. The recombinant <em>Ng</em>HisD lost 85 % activity in the presence of 1 mM 1,10-phenantroline. Our study not only establishes a foundation for investigating the physiological function of <em>Ng</em>HisD <em>in vivo</em> but also uncovers the absence of the canonical histidine biosynthesis pathway in <em>Neisseria cinerea</em>.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106824"},"PeriodicalIF":1.2,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186531","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 : 2026-01-01Epub Date: 2025-10-08DOI: 10.1016/j.pep.2025.106826
Taiji Oyama, Satoko Suzuki, Ken-ichi Akao
Circular dichroism (CD) spectroscopy is a rapid and versatile method for assessing the higher-order structures of proteins and peptides. Far-UV CD enables quantitative evaluation of secondary structures, while near-UV CD provides sensitive fingerprints of tertiary organization. With advances in recombinant protein expression, CD spectroscopy has become widely used for the characterization of novel generated proteins. This review focuses on the applications of CD spectroscopy in protein engineering and pharmaceutical sciences. Examples include strategies for reliable measurements with limited sample quantities and quality assessments such as lot-to-lot comparisons and biosimilar evaluations. CD spectroscopy also serves as a valuable tool for detecting conformational changes associated with protein–protein and protein–drug interactions, as well as for evaluating proteins and peptides in membrane-mimetic environments. Obtaining reliable CD spectra requires careful selection of buffers, water-miscible solvents, and excipients. Here, we summarize their properties and propose practical criteria for their selection to ensure high-quality CD measurements.
{"title":"Circular dichroism spectroscopy in protein engineering and pharmaceutical development: Applications in structural characterization and quality assessment","authors":"Taiji Oyama, Satoko Suzuki, Ken-ichi Akao","doi":"10.1016/j.pep.2025.106826","DOIUrl":"10.1016/j.pep.2025.106826","url":null,"abstract":"<div><div>Circular dichroism (CD) spectroscopy is a rapid and versatile method for assessing the higher-order structures of proteins and peptides. Far-UV CD enables quantitative evaluation of secondary structures, while near-UV CD provides sensitive fingerprints of tertiary organization. With advances in recombinant protein expression, CD spectroscopy has become widely used for the characterization of novel generated proteins. This review focuses on the applications of CD spectroscopy in protein engineering and pharmaceutical sciences. Examples include strategies for reliable measurements with limited sample quantities and quality assessments such as lot-to-lot comparisons and biosimilar evaluations. CD spectroscopy also serves as a valuable tool for detecting conformational changes associated with protein–protein and protein–drug interactions, as well as for evaluating proteins and peptides in membrane-mimetic environments. Obtaining reliable CD spectra requires careful selection of buffers, water-miscible solvents, and excipients. Here, we summarize their properties and propose practical criteria for their selection to ensure high-quality CD measurements.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106826"},"PeriodicalIF":1.2,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259018","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 : 2026-01-01Epub Date: 2025-07-25DOI: 10.1016/j.pep.2025.106769
Swapna G. Naik , Dong Keun Rhee , Steven Hockman , Faiyaz Ahmad Khan , Arun Samidurai , Babak Sabouri , Noel Carter , Vincent C. Manganiello
Phosphodiesterase 3 enzymes (PDE3) play important roles in the regulation of adipocyte lipolysis and cardiac contractility by hydrolyzing cAMP and cGMP. This study reports the cloning, expression, and purification of recombinant Caenorhabditis elegans phosphodiesterase 3 (CEPDE3) isoforms, using a cold shock-based technique. The two closely related isoforms of the CEPDE3 gene (isoform F and isoform A) were cloned into the pGEX-6P-1 vector and expressed in E. coli as fusion proteins with a glutathione-S transferase (GST) tag at their amino terminus and purified by affinity chromatography using a glutathione Sepharose column. To optimize expression and recovery of soluble CEPDE3 protein from E. coli, we applied a “cold shock” technique at 4 °C following IPTG induction. Our findings suggest improved protein expression using an N-terminal GST tag instead of a C-terminal 6-histidine (6His) tag. Exposure of GST-tagged CEPDE3 to cold shock improved the protein solubility of CEPDE3 isoforms recovered by affinity chromatography. Studying CEPDE3 expression in C. elegans may enable us to understand the structure-function relationship and help crystallize the proteins to identify its catalytic pocket, contributing to the design of more effective small modulators of PDE3 catalytic activity.
{"title":"Validation of cold shock-based technique for purification of recombinant C. elegans Phosphodiesterase 3 protein expressed in E. coli","authors":"Swapna G. Naik , Dong Keun Rhee , Steven Hockman , Faiyaz Ahmad Khan , Arun Samidurai , Babak Sabouri , Noel Carter , Vincent C. Manganiello","doi":"10.1016/j.pep.2025.106769","DOIUrl":"10.1016/j.pep.2025.106769","url":null,"abstract":"<div><div>Phosphodiesterase 3 enzymes (PDE3) play important roles in the regulation of adipocyte lipolysis and cardiac contractility by hydrolyzing cAMP and cGMP. This study reports the cloning, expression, and purification of recombinant <em>Caenorhabditis elegans</em> phosphodiesterase 3 (CEPDE3) isoforms, using a cold shock-based technique. The two closely related isoforms of the <em>CEPDE3</em> gene (isoform F and isoform A) were cloned into the pGEX-6P-1 vector and expressed in <em>E. coli</em> as fusion proteins with a glutathione-S transferase (GST) tag at their amino terminus and purified by affinity chromatography using a glutathione Sepharose column. To optimize expression and recovery of soluble CEPDE3 protein from <em>E. coli</em>, we applied a “cold shock” technique at 4 °C following IPTG induction. Our findings suggest improved protein expression using an N-terminal GST tag instead of a C-terminal 6-histidine (6His) tag. Exposure of GST-tagged CEPDE3 to cold shock improved the protein solubility of CEPDE3 isoforms recovered by affinity chromatography. Studying CEPDE3 expression in <em>C. elegans</em> may enable us to understand the structure-function relationship and help crystallize the proteins to identify its catalytic pocket, contributing to the design of more effective small modulators of PDE3 catalytic activity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106769"},"PeriodicalIF":1.2,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732922","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 : 2026-01-01Epub Date: 2025-09-25DOI: 10.1016/j.pep.2025.106823
Gaoya Yuan, Meng Qu, Yifeng Li
Half-antibody is a common byproduct associated with the production of asymmetric bispecific antibody (bsAb). We previously demonstrated that Protein A column chromatography can effectively separate this byproduct from the bsAb product, as these two species exhibit different binding valencies (half-antibody and intact bsAb contain one and two Protein A binding sites, respectively). Specifically, half-antibody, which binds weaker, can be selectively removed by an appropriate wash prior to elution of the target bsAb. However, the performance of this wash step is sensitive to the loading density, making the process unrobust to variations in harvest titer. In the current study, we demonstrate that Sartobind Rapid A Protein A membrane effectively reduces half-antibody from approximately 16.5% (in the load) to 0.1% (in the eluate). In addition, using Protein A membrane also improves process robustness by avoiding the impact of loading density variation on performance. Thus, for bsAb purification where half-antibody removal is required, Protein A membrane is a better choice than resin-based Protein A column.
半抗体是与不对称双特异性抗体（bsAb）产生相关的常见副产物。我们之前证明了蛋白A柱层析可以有效地将这种副产物从bsAb产物中分离出来，因为这两个物种具有不同的结合价（半抗体和完整的bsAb分别含有一个和两个蛋白A结合位点）。具体来说，结合较弱的半抗体可以在洗脱目标bsAb之前通过适当的洗涤选择性地去除。然而，该洗涤步骤的性能对装载密度很敏感，使得该过程对收获滴度的变化不稳定。在目前的研究中，我们证明Sartobind Rapid A Protein A膜有效地将半抗体从大约16.5%（负载）降低到0.1%（洗脱液）。此外，使用蛋白A膜还可以避免负载密度变化对性能的影响，从而提高工艺稳健性。因此，对于需要去除半抗体的bsAb纯化，蛋白A膜是比基于树脂的蛋白A柱更好的选择。
{"title":"Effective and robust separation of half-antibody byproduct in bispecific antibody purification by Sartobind Rapid A Protein A membrane chromatography","authors":"Gaoya Yuan, Meng Qu, Yifeng Li","doi":"10.1016/j.pep.2025.106823","DOIUrl":"10.1016/j.pep.2025.106823","url":null,"abstract":"<div><div>Half-antibody is a common byproduct associated with the production of asymmetric bispecific antibody (bsAb). We previously demonstrated that Protein A column chromatography can effectively separate this byproduct from the bsAb product, as these two species exhibit different binding valencies (half-antibody and intact bsAb contain one and two Protein A binding sites, respectively). Specifically, half-antibody, which binds weaker, can be selectively removed by an appropriate wash prior to elution of the target bsAb. However, the performance of this wash step is sensitive to the loading density, making the process unrobust to variations in harvest titer. In the current study, we demonstrate that Sartobind Rapid A Protein A membrane effectively reduces half-antibody from approximately 16.5% (in the load) to 0.1% (in the eluate). In addition, using Protein A membrane also improves process robustness by avoiding the impact of loading density variation on performance. Thus, for bsAb purification where half-antibody removal is required, Protein A membrane is a better choice than resin-based Protein A column.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106823"},"PeriodicalIF":1.2,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145182205","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: 2025-09-04DOI: 10.1016/j.pep.2025.106810
Hongxia Li, Xiong Wang, Xinye Wang, Zhang Zhang, Linmin Ran
Aldehyde dehydrogenase 2 (ALDH2) plays a critical role in ethanol metabolism by converting toxic acetaldehyde to acetate. To investigate its functional mechanisms and potential therapeutic applications for alcohol-related diseases, heterologous expression of ALDH2 is essential. However, ALDH2 often forms inclusion bodies when expressed in Escherichia coli. In this work, the solubility of ALDH2 was enhanced by systematic optimization of expression conditions using IPTG and lactose as respective inducers. Under optimized conditions, the media yield of ALDH2 induced by IPTG and lactose reached 44.5 ± 1.3 and 48.7 ± 1.2 μg/mL respectively, representing 7.1- and 7.8-fold improvements over unoptimized conditions. Enzymatic characterization revealed that purified ALDH2 exhibited optimal activity of 9.7 U/mL at 37 °C and pH 8.0. This research demonstrates that optimizing expression conditions is an effective strategy to enhance the solubility of recombinant enzymes, while providing a practical solution for other enzymes prone to inclusion body formation.
{"title":"High-level soluble expression of human aldehyde dehydrogenase 2 in Escherichia coli achieved through lactose-mediated induction optimization","authors":"Hongxia Li, Xiong Wang, Xinye Wang, Zhang Zhang, Linmin Ran","doi":"10.1016/j.pep.2025.106810","DOIUrl":"10.1016/j.pep.2025.106810","url":null,"abstract":"<div><div>Aldehyde dehydrogenase 2 (ALDH2) plays a critical role in ethanol metabolism by converting toxic acetaldehyde to acetate. To investigate its functional mechanisms and potential therapeutic applications for alcohol-related diseases, heterologous expression of ALDH2 is essential. However, ALDH2 often forms inclusion bodies when expressed in <em>Escherichia coli</em>. In this work, the solubility of ALDH2 was enhanced by systematic optimization of expression conditions using IPTG and lactose as respective inducers. Under optimized conditions, the media yield of ALDH2 induced by IPTG and lactose reached 44.5 ± 1.3 and 48.7 ± 1.2 μg/mL respectively, representing 7.1- and 7.8-fold improvements over unoptimized conditions. Enzymatic characterization revealed that purified ALDH2 exhibited optimal activity of 9.7 U/mL at 37 °C and pH 8.0. This research demonstrates that optimizing expression conditions is an effective strategy to enhance the solubility of recombinant enzymes, while providing a practical solution for other enzymes prone to inclusion body formation.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106810"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145004120","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.2025.106789
Rahul Singh , Ravindra D. Makde
Heme, a toxic by-product of Plasmodium's proteolytic digestion of host hemoglobin, is detoxified by the malaria parasite through its conversion into hemozoin (Hz)—the malaria pigment. This detoxification pathway is a key target for many antimalarial drugs, which aim to induce heme-mediated toxicity to the parasite. The Heme Detoxification Protein (HDP) plays a central role in heme-to-Hz transformation; however, its precise mechanism remains unclear, largely due to the absence of successful recombinant expression in a native, soluble form.
In this study, we aimed to express HDP recombinantly in its native soluble state using an E. coli-based system. A range of strategies were employed, including expression of orthologs, consensus sequence design, fusion to solubility-enhancing partners, co-expression with molecular chaperones, and extensive construct optimization through N-terminal truncations. Despite extensive efforts, most recombinant HDP constructs were either insoluble or formed soluble aggregates. Notably, only a single construct—with a 44-residue N-terminal truncation and a C-terminal 6-His tag (HDPpf-C10)—was successfully expressed in a soluble form.
Surprisingly, HDPpf-C10, although retaining domains implicated in heme binding and transformation, exhibited no detectable heme-to-Hz transformation activity. This finding highlights the essential role of the flexible-unstructured N-terminal region in mediating both heme binding and its subsequent conversion to Hz, providing new insights into HDP function and guiding future structural and mechanistic studies.
{"title":"Recombinant expression and purification of Plasmodium heme detoxification protein in E. coli: Challenges and discoveries","authors":"Rahul Singh , Ravindra D. Makde","doi":"10.1016/j.pep.2025.106789","DOIUrl":"10.1016/j.pep.2025.106789","url":null,"abstract":"<div><div>Heme, a toxic by-product of <em>Plasmodium</em>'s proteolytic digestion of host hemoglobin, is detoxified by the malaria parasite through its conversion into hemozoin (Hz)—the malaria pigment. This detoxification pathway is a key target for many antimalarial drugs, which aim to induce heme-mediated toxicity to the parasite. The Heme Detoxification Protein (HDP) plays a central role in heme-to-Hz transformation; however, its precise mechanism remains unclear, largely due to the absence of successful recombinant expression in a native, soluble form.</div><div>In this study, we aimed to express HDP recombinantly in its native soluble state using an <em>E. coli</em>-based system. A range of strategies were employed, including expression of orthologs, consensus sequence design, fusion to solubility-enhancing partners, co-expression with molecular chaperones, and extensive construct optimization through N-terminal truncations. Despite extensive efforts, most recombinant HDP constructs were either insoluble or formed soluble aggregates. Notably, only a single construct—with a 44-residue N-terminal truncation and a C-terminal 6-His tag (<em>HDPpf-C10</em>)—was successfully expressed in a soluble form.</div><div>Surprisingly, HDPpf-C10, although retaining domains implicated in heme binding and transformation, exhibited no detectable heme-to-Hz transformation activity. This finding highlights the essential role of the flexible-unstructured N-terminal region in mediating both heme binding and its subsequent conversion to Hz, providing new insights into HDP function and guiding future structural and mechanistic studies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106789"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144785126","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: 2025-09-05DOI: 10.1016/j.pep.2025.106811
Thanh-Hoa T. Tran , Trung-Duc Nguyen , Ngoc-Nam Phan , Hang T. Ngo , Phuc-Loc Nguyen Do , Phan-Anh Le , Nho-Thai Dinh , Tuan-Nghia Phan , Hong-Loan T. Nguyen
The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6×His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (y = 725.29x − 52.356; R2 = 0.998) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a Km of 22.01 ± 3.5 μM, kcat of 0.021 s-1, and catalytic efficiency (kcat/Km) of 946 M-1.s-1. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC50 of 0.88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.
{"title":"A magnetic bead-based fluorescent substrate for sensitive assay of SARS-CoV-2 3C-like protease activity","authors":"Thanh-Hoa T. Tran , Trung-Duc Nguyen , Ngoc-Nam Phan , Hang T. Ngo , Phuc-Loc Nguyen Do , Phan-Anh Le , Nho-Thai Dinh , Tuan-Nghia Phan , Hong-Loan T. Nguyen","doi":"10.1016/j.pep.2025.106811","DOIUrl":"10.1016/j.pep.2025.106811","url":null,"abstract":"<div><div>The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6×His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (<em>y</em> = <em>725.29x</em> − <em>52.356</em>; <em>R</em><sup><em>2</em></sup> = <em>0.998</em>) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a K<sub>m</sub> of 22.01 ± 3.5 μM, k<sub>cat</sub> of 0.021 s<sup>-</sup><sup>1</sup>, and catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub>) of 946 M<sup>-</sup><sup>1</sup><sup>.</sup>s<sup>-</sup><sup>1</sup>. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC<sub>50</sub> of 0.88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106811"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145016039","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}
Human interleukin-2 (hIL-2) serves as a crucial cytokine in the treatment of cancer and autoimmune disorders. Nevertheless, the advancement of research and clinical applications involving this cytokine has been hindered by the constraints associated with the production of recombinant human interleukin-2 (rhIL-2). This study presents a scalable and robust purification protocol for rhIL-2 derived from inclusion bodies (IBs) in Escherichia coli. Our results indicate that microfiltration-based method could improve the purity of the denatured IBs effectively, and various refolding conditions were assessed to improve the recovery of refolded rhIL-2, resulting in an increase in the refolding yield from 15 % to 45 %. Subsequently, purification through three-column chromatography could refine the refolded rhIL-2 efficiently. Ultimately, the robustness of the purification process is substantiated by three consecutive scale-up experiments, achieving a productivity of 4 mg rhIL-2/g cell pellets, alongside high product purity and significant product activity.
{"title":"High-efficient refolding and purification of recombinant human interleukin-2 from inclusion bodies","authors":"Fei Wang , Yuming Fang , Jiawei Yu , Xinyi Zhao, Yuxiao Liu, Xiaoran Jing, Jiayu Wang, Shanshan Wang, Shuo Wang, Junjun Jiang, Sheng Zhang","doi":"10.1016/j.pep.2025.106806","DOIUrl":"10.1016/j.pep.2025.106806","url":null,"abstract":"<div><div>Human interleukin-2 (hIL-2) serves as a crucial cytokine in the treatment of cancer and autoimmune disorders. Nevertheless, the advancement of research and clinical applications involving this cytokine has been hindered by the constraints associated with the production of recombinant human interleukin-2 (rhIL-2). This study presents a scalable and robust purification protocol for rhIL-2 derived from inclusion bodies (IBs) in <em>Escherichia coli</em>. Our results indicate that microfiltration-based method could improve the purity of the denatured IBs effectively, and various refolding conditions were assessed to improve the recovery of refolded rhIL-2, resulting in an increase in the refolding yield from 15 % to 45 %. Subsequently, purification through three-column chromatography could refine the refolded rhIL-2 efficiently. Ultimately, the robustness of the purification process is substantiated by three consecutive scale-up experiments, achieving a productivity of 4 mg rhIL-2/g cell pellets, alongside high product purity and significant product activity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106806"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144918028","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: 2025-08-27DOI: 10.1016/j.pep.2025.106807
Muhammad Naeem , Weihua Zhao , Tengjian Wen , Rong Han , Xuemeng Shan , Anran Xu , Lingxia Zhao
Plant-based expression systems offer a promising platform for producing therapeutic glycoproteins with human-compatible glycosylation patterns. This study aimed to engineer tobacco plants (Nicotiana tabacum cv. Yunyan 87) to modify glycosylation pathways for the production of glycoproteins with reduced immunogenicity, enhancing their potential for therapeutic applications. To achieve this, a 1257 bp fragment of the human β-1,4-galactosyltransferase (GALT) gene was cloned into the pHB vector and introduced into tobacco via Agrobacterium-mediated transformation. Four GALT-OE lines (13#, 18#, 22# and 30#) were generated which showed significantly higher GALT expression, especially GALT-OE 30# which showed a 4.5-fold increase over wild-type (WT). Moreover, Western-blot and ELISA analyses showed that protein expression in galt13#, and galt30# was also increased. Triple mutants were generated by crossing the GALT-OE 30# line with previously developed double mutants β-1,2-xylosyltransferase (CXT1P-RNAi) and α-1,3-fucosyltransferase (FUT4-RNAi), which showed a 70 % and 80 % reduction in CXT1P and FUT4 expression levels, respectively. The generated triple mutants (cfG028, cfG031, and cfG039) showed a 3.8-fold increase in GALT expression, and corresponding glycoprotein modifications at the protein level. This study establishes a foundation for the large-scale production of low-immunogenic recombinant glycoproteins with enhanced therapeutic efficacy using a tobacco-based system.
{"title":"Creating a triple mutant tobacco chassis with altered cfG expression for the production of humanized therapeutic protein","authors":"Muhammad Naeem , Weihua Zhao , Tengjian Wen , Rong Han , Xuemeng Shan , Anran Xu , Lingxia Zhao","doi":"10.1016/j.pep.2025.106807","DOIUrl":"10.1016/j.pep.2025.106807","url":null,"abstract":"<div><div>Plant-based expression systems offer a promising platform for producing therapeutic glycoproteins with human-compatible glycosylation patterns. This study aimed to engineer tobacco plants (<em>Nicotiana tabacum</em> cv. Yunyan 87) to modify glycosylation pathways for the production of glycoproteins with reduced immunogenicity, enhancing their potential for therapeutic applications. To achieve this, a 1257 bp fragment of the human <em>β-1,4-galactosyltransferase</em> (<em>GALT</em>) gene was cloned into the <em>pHB</em> vector and introduced into tobacco <em>via Agrobacterium</em>-mediated transformation. Four <em>GALT</em>-OE lines (<em>13</em><sup><em>#</em></sup><em>, 18</em><sup><em>#</em></sup><em>, 22</em><sup><em>#</em></sup> and <em>30</em><sup><em>#</em></sup>) were generated which showed significantly higher <em>GALT</em> expression, especially <em>GALT-</em>OE <em>30</em><sup><em>#</em></sup> which showed a 4.5-fold increase over wild-type (WT). Moreover, Western-blot and ELISA analyses showed that protein expression in <em>galt13</em><sup><em>#</em></sup>, and <em>galt30</em><sup><em>#</em></sup> was also increased. Triple mutants were generated by crossing the <em>GALT-</em>OE 30<sup>#</sup> line with previously developed double mutants <em>β-1,2-xylosyltransferase</em> (<em>CXT1P</em>-RNAi) and <em>α-1,3-fucosyltransferase</em> (<em>FUT4</em>-RNAi), which showed a 70 % and 80 % reduction in <em>CXT1P</em> and <em>FUT4</em> expression levels, respectively. The generated triple mutants (<em>cfG028, cfG031,</em> and <em>cfG039</em>) showed a 3.8-fold increase in <em>GALT</em> expression, and corresponding glycoprotein modifications at the protein level. This study establishes a foundation for the large-scale production of low-immunogenic recombinant glycoproteins with enhanced therapeutic efficacy using a tobacco-based system.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106807"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966316","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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