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":"2025-08-23","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 : 2025-08-23DOI: 10.1016/j.pep.2025.106805
Subhankar Metya , Shoaib Haidar , Anurag S. Rathore
Single-domain antibodies, known as nanobodies, have arisen as an exciting class of biomolecules with applications ranging from diagnostics to numerous therapies. Due to their small size, enhanced stability, and reduced production complexity compared to traditional monoclonal antibodies, nanobodies present an economically attractive option with considerable commercial promise. In this paper, we describe a robust, efficient, and cost-effective downstream purification platform for production of nanobody-based biotherapeutics produced in Escherichia coli (E. coli). A peptide fused with the nanobody has been taken as a model. Our process utilizes periplasmic extraction to obtain the soluble product, followed by a mild acid precipitation step to remove impurities, and a single multimodal chromatography step, with an overall yield of 82.6 % and a purity exceeding 95 %. An extensive cost analysis indicated a production cost of about $17.7 per gram, far cheaper than the conventional mAb-based biopharmaceuticals. The proposed platform can facilitate commercialization of therapeutic nanobodies.
{"title":"Efficient and economical purification platform for production of therapeutic nanobodies","authors":"Subhankar Metya , Shoaib Haidar , Anurag S. Rathore","doi":"10.1016/j.pep.2025.106805","DOIUrl":"10.1016/j.pep.2025.106805","url":null,"abstract":"<div><div>Single-domain antibodies, known as nanobodies, have arisen as an exciting class of biomolecules with applications ranging from diagnostics to numerous therapies. Due to their small size, enhanced stability, and reduced production complexity compared to traditional monoclonal antibodies, nanobodies present an economically attractive option with considerable commercial promise. In this paper, we describe a robust, efficient, and cost-effective downstream purification platform for production of nanobody-based biotherapeutics produced in <em>Escherichia coli</em> (<em>E. coli)</em>. A peptide fused with the nanobody has been taken as a model. Our process utilizes periplasmic extraction to obtain the soluble product, followed by a mild acid precipitation step to remove impurities, and a single multimodal chromatography step, with an overall yield of 82.6 % and a purity exceeding 95 %. An extensive cost analysis indicated a production cost of about $17.7 per gram, far cheaper than the conventional mAb-based biopharmaceuticals. The proposed platform can facilitate commercialization of therapeutic nanobodies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106805"},"PeriodicalIF":1.2,"publicationDate":"2025-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144893767","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 : 2025-08-20DOI: 10.1016/j.pep.2025.106804
Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan
Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant Ananas comosus. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) Hac1 on folding and secretion of recombinant stem bromelain in Pichia pastoris. Stem bromelain gene (BL) from Ananas comosus was codon optimized and expressed in the Pichia pastoris X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, Hac1p was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when HAC1 was co-expressed with stem bromelain (BL). Fed batch studies were performed for both pGAPαBL1 and pGAPBLHAC1 clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones like Kar2p, Sec 63, Pdi, Cne1. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies Pichia pastoris as a robust platform to produce stem bromelain for various industrial applications.
{"title":"Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P","authors":"Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan","doi":"10.1016/j.pep.2025.106804","DOIUrl":"10.1016/j.pep.2025.106804","url":null,"abstract":"<div><div>Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant <em>Ananas comosus</em>. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) <em>Hac1</em> on folding and secretion of recombinant stem bromelain in <em>Pichia pastoris.</em> Stem bromelain gene (BL) from <em>Ananas comosus</em> was codon optimized and expressed in the <em>Pichia pastoris</em> X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, <em>Hac1p</em> was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when <em>HAC1</em> was co-expressed with stem bromelain (BL). Fed batch studies were performed for both <em>pGAPαBL1</em> and <em>pGAPBLHAC1</em> clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones <em>like Kar2p, Sec 63, Pdi, Cne1</em>. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies <em>Pichia pastoris</em> as a robust platform to produce stem bromelain for various industrial applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106804"},"PeriodicalIF":1.2,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144918027","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 : 2025-08-18DOI: 10.1016/j.pep.2025.106802
Shusei Mizushima
A number of methods are used to produce recombinant proteins, and animal bioreactors have emerged as transgenic systems. Animal bioreactors have the potential to reduce production costs and improve efficiency, thereby providing recombinant proteins that are important for therapeutic applications. Various species, such as goats, cattle, and rabbits, have been genetically modified to serve as bioreactors. Since poultry hens produce more than 300 eggs per year, their eggs may also be used as an efficient platform for the production of protein pharmaceuticals. In addition to traditional viral infection of the blastoderm, the recent development of genome-editing technologies for cultured primordial germ cells has enabled the establishment of transgenic chicken lines via germline chimera production systems. We established a new genome-editing technology combined with the intracytoplasmic sperm injection (ICSI) method that has the potential to produce transgenic quail very efficiently in one generation. The avian ICSI technique and recent advances in genome editing are discussed herein.
{"title":"Intracytoplasmic sperm injection-mediated strategy for the production of transgenic poultry as a bioreactor","authors":"Shusei Mizushima","doi":"10.1016/j.pep.2025.106802","DOIUrl":"10.1016/j.pep.2025.106802","url":null,"abstract":"<div><div>A number of methods are used to produce recombinant proteins, and animal bioreactors have emerged as transgenic systems. Animal bioreactors have the potential to reduce production costs and improve efficiency, thereby providing recombinant proteins that are important for therapeutic applications. Various species, such as goats, cattle, and rabbits, have been genetically modified to serve as bioreactors. Since poultry hens produce more than 300 eggs per year, their eggs may also be used as an efficient platform for the production of protein pharmaceuticals. In addition to traditional viral infection of the blastoderm, the recent development of genome-editing technologies for cultured primordial germ cells has enabled the establishment of transgenic chicken lines via germline chimera production systems. We established a new genome-editing technology combined with the intracytoplasmic sperm injection (ICSI) method that has the potential to produce transgenic quail very efficiently in one generation. The avian ICSI technique and recent advances in genome editing are discussed herein.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106802"},"PeriodicalIF":1.2,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144863440","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 : 2025-08-18DOI: 10.1016/j.pep.2025.106803
Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski
Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (α1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: Trichoplusia ni insect cells (High Five) and human embryonic kidney cells (Expi293F™). The High Five cells generate short oligomannose structures, while the Expi293F™ cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F™-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F™ cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F™-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts.
{"title":"Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation","authors":"Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski","doi":"10.1016/j.pep.2025.106803","DOIUrl":"10.1016/j.pep.2025.106803","url":null,"abstract":"<div><div>Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (α1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: <em>Trichoplusia ni</em> insect cells (High Five) and human embryonic kidney cells (Expi293F™). The High Five cells generate short oligomannose structures, while the Expi293F™ cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F™-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F™ cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F™-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106803"},"PeriodicalIF":1.2,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144912957","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 : 2025-08-15DOI: 10.1016/j.pep.2025.106801
Katerina Jiraskova, Jakub Ptacek, Kristyna Vydra Bousova, Jiri Vondrasek
The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered IsPETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management.
{"title":"Toward reproducible PETase research: A standardized workflow for reliable enzyme production and comparison","authors":"Katerina Jiraskova, Jakub Ptacek, Kristyna Vydra Bousova, Jiri Vondrasek","doi":"10.1016/j.pep.2025.106801","DOIUrl":"10.1016/j.pep.2025.106801","url":null,"abstract":"<div><div>The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered <em>Is</em>PETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106801"},"PeriodicalIF":1.2,"publicationDate":"2025-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144862366","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 : 2025-08-05DOI: 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":"2025-08-05","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 : 2025-08-05DOI: 10.1016/j.pep.2025.106790
Jafar Nikzad , Kimia Kalantari Khandani , Yeganeh Talebkhan , Fatemeh Zandi , Samira Komijani , Ahmad Adeli
Nearly four decades have passed since the discovery of protein-L. Its exceptional ability in binding to the kappa light chain of immunoglobulins makes it a suitable candidate for the purification of certain biotherapeutics, particularly antibody fragments. Efforts have focused on improving its recovery and dynamic binding capacity. Among various strategies, ligand multimerization has shown significant potential in developing more efficient and cost-effective resins. This study employed a multimerization approach to create new recombinant protein-L-based ligands and compare their performance to the commercially available alternatives. Dynamic binding capacity studies revealed that the engineered ProL6 and ProL8 resins exhibited higher binding capacities than the ProL4 and the commercial Capto-L resin. Furthermore, the recovery rates of Fab antibody fragments from bacterial lysates using ProL6, ProL8, and the commercial resin were 92.8, 94.4, and 94.6 %, respectively, comparable to those of the ProL4 resin. SDS-PAGE analysis confirmed the purity of the proteins eluted from all tested resins, aligned with the results of ProL4 resin. Additionally, it evaluated the 100 % specificity of ProL6, ProL8, and the commercial resins.
{"title":"Ligand multimerization effects on binding efficiency of Protein L affinity chromatography resins","authors":"Jafar Nikzad , Kimia Kalantari Khandani , Yeganeh Talebkhan , Fatemeh Zandi , Samira Komijani , Ahmad Adeli","doi":"10.1016/j.pep.2025.106790","DOIUrl":"10.1016/j.pep.2025.106790","url":null,"abstract":"<div><div>Nearly four decades have passed since the discovery of protein-L. Its exceptional ability in binding to the kappa light chain of immunoglobulins makes it a suitable candidate for the purification of certain biotherapeutics, particularly antibody fragments. Efforts have focused on improving its recovery and dynamic binding capacity. Among various strategies, ligand multimerization has shown significant potential in developing more efficient and cost-effective resins. This study employed a multimerization approach to create new recombinant protein-L-based ligands and compare their performance to the commercially available alternatives. Dynamic binding capacity studies revealed that the engineered ProL6 and ProL8 resins exhibited higher binding capacities than the ProL4 and the commercial Capto-L resin. Furthermore, the recovery rates of Fab antibody fragments from bacterial lysates using ProL6, ProL8, and the commercial resin were 92.8, 94.4, and 94.6 %, respectively, comparable to those of the ProL4 resin. SDS-PAGE analysis confirmed the purity of the proteins eluted from all tested resins, aligned with the results of ProL4 resin. Additionally, it evaluated the 100 % specificity of ProL6, ProL8, and the commercial resins.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106790"},"PeriodicalIF":1.2,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144770612","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 : 2025-07-31DOI: 10.1016/j.pep.2025.106788
Manami Morimoto , Daisuke Saito
Since Swift's discovery of primordial germ cells (PGCs) within the vasculature of chicken embryos in 1914, significant progress has been made in uncovering the origins, migratory pathways, and molecular characteristics of avian PGCs. Recent advances in this field have been accelerated by two synergistic factors: the establishment of robust culture systems that enable efficient genetic manipulation of chicken PGCs, and the inherent suitability of avian embryos for cell transplantation and live imaging. Together, these features have positioned avian PGCs as a powerful system for investigating not only cell migration but also long-standing mysteries in germ cell biology. Furthermore, this system offers a unique platform for dissecting the cellular and molecular mechanisms underlying diseases such as cancer metastasis and germ cell tumors. In this review, we revisit key historical milestones in avian PGC research, explore current knowledge of their migratory regulation, and highlight future directions with potential impact across cell biology, developmental biology, and disease modeling.
{"title":"Avian primordial germ cell migration: History, mechanisms, applications, and unanswered questions","authors":"Manami Morimoto , Daisuke Saito","doi":"10.1016/j.pep.2025.106788","DOIUrl":"10.1016/j.pep.2025.106788","url":null,"abstract":"<div><div>Since Swift's discovery of primordial germ cells (PGCs) within the vasculature of chicken embryos in 1914, significant progress has been made in uncovering the origins, migratory pathways, and molecular characteristics of avian PGCs. Recent advances in this field have been accelerated by two synergistic factors: the establishment of robust culture systems that enable efficient genetic manipulation of chicken PGCs, and the inherent suitability of avian embryos for cell transplantation and live imaging. Together, these features have positioned avian PGCs as a powerful system for investigating not only cell migration but also long-standing mysteries in germ cell biology. Furthermore, this system offers a unique platform for dissecting the cellular and molecular mechanisms underlying diseases such as cancer metastasis and germ cell tumors. In this review, we revisit key historical milestones in avian PGC research, explore current knowledge of their migratory regulation, and highlight future directions with potential impact across cell biology, developmental biology, and disease modeling.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106788"},"PeriodicalIF":1.2,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768957","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 : 2025-07-30DOI: 10.1016/j.pep.2025.106786
Young Su Kim , Hye-Jeong Lee , Mi-Reu Kim , Hwabong Jeong , Young Pil Kim , Jung-Ho Park , Jungoh Ahn
Human plasma gelsolin (pGSN) is an 83 kDa actin-binding protein involved in cytoskeletal remodeling, inflammation, and host defense. Its clinical relevance as a biomarker and potential therapeutic agent, particularly in conditions like sepsis, acute respiratory distress syndrome (ARDS), and cystic fibrosis, has driven interest in scalable recombinant expression. However, high-yield production of functionally active gelsolin is hindered by its complex structure and folding requirements. To address this, we developed a scalable, high-yield bacterial expression system that achieves among the highest reported levels of functional recombinant human gelsolin (rGelsolin) using a GST-fusion strategy incorporating a tobacco etch virus (TEV) protease cleavage site, optimized for solubility and downstream processing. High-density fed-batch fermentation in E. coli yielded 5.0 g/L of soluble protein. Following a three-step purification process with removal of the GST tag, 2.1 g/L of tag-free, high-purity rGelsolin with >95 % purity was obtained. Structural characterization by circular dichroism spectroscopy confirmed that rGelsolin adopted a native-like secondary structure and exhibited thermal stability (Tm ∼59 °C). Correct processing of the recombinant protein was verified by N- and C-terminal sequencing. Functional assays demonstrated that rGelsolin bound to and severed actin filaments in a calcium-dependent manner, similar to native plasma gelsolin. These findings demonstrate a scalable, cost-effective platform for producing bioactive rGelsolin in E. coli, with structural and functional features comparable to native pGSN, supporting its potential utility in diagnostic, therapeutic, and structural applications in the context of acute and chronic inflammatory diseases.
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