Pub Date : 2025-12-01Epub 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":"2025-12-01","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 : 2025-12-01Epub 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}
Pub Date : 2025-12-01Epub 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":"2025-12-01","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 : 2025-12-01Epub 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":"2025-12-01","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 : 2025-12-01Epub Date: 2025-08-05DOI: 10.1016/j.pep.2025.106787
Tetsuya Wakabayashi, Taichi Kuramochi
Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.
{"title":"Discovery and development of bispecific antibodies.","authors":"Tetsuya Wakabayashi, Taichi Kuramochi","doi":"10.1016/j.pep.2025.106787","DOIUrl":"10.1016/j.pep.2025.106787","url":null,"abstract":"<p><p>Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106787"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144776016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub 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":"2025-12-01","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 : 2025-12-01Epub Date: 2025-08-31DOI: 10.1016/j.pep.2025.106809
Leheng Chen , Dawei Fu
The DNA polymerase from Thermococcus kodakarensis KOD1 (KOD) is widely utilized in polymerase chain reaction (PCR) due to its high processivity and fidelity. However, like many other B-family DNA polymerases, it faces limitations in extension efficiency, amplicon length, and resistance to PCR inhibitors. In order to further enhance its capability, novel mutants were engineered by fusing a 7 kDa nonspecific double-stranded DNA (dsDNA)-binding protein from Sulfolobus tokodaii (Sto7d) to the C-terminus of KOD via distinct peptide linkers, resulting in a set of KOD-Sto7d polymerase variants. These constructs were expressed, purified, and characterized. Among the variants, KOD-GT4G-Sto7d exhibited the best PCR performance and was selected as the representative variant for subsequent assays. Compared with wild-type KOD (KOD-WT), KOD-Sto7d demonstrated significantly improved extension efficiency that successfully amplified 7 kb targets with only 10 s elongation time, increased salt tolerance up to 120 mM NaCl for 2 kb targets, and an improved capacity to amplify long DNA fragments up to 10 kb within 4 min. In comparison with a commercially available KOD mutant fused to a dsDNA-binding protein (Sso7d from Saccharolobus solfataricus) at its C-terminus (KOD-Sso7d), KOD-Sto7d demonstrated greater salt tolerance and sensitivity. These results suggest that KOD-Sto7d is a robust polymerase suitable for time-saving and high-demanding PCR.
{"title":"Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d","authors":"Leheng Chen , Dawei Fu","doi":"10.1016/j.pep.2025.106809","DOIUrl":"10.1016/j.pep.2025.106809","url":null,"abstract":"<div><div>The DNA polymerase from <em>Thermococcus kodakarensis</em> KOD1 (KOD) is widely utilized in polymerase chain reaction (PCR) due to its high processivity and fidelity. However, like many other B-family DNA polymerases, it faces limitations in extension efficiency, amplicon length, and resistance to PCR inhibitors. In order to further enhance its capability, novel mutants were engineered by fusing a 7 kDa nonspecific double-stranded DNA (dsDNA)-binding protein from <em>Sulfolobus tokodaii</em> (Sto7d) to the C-terminus of KOD via distinct peptide linkers, resulting in a set of KOD-Sto7d polymerase variants. These constructs were expressed, purified, and characterized. Among the variants, KOD-GT4G-Sto7d exhibited the best PCR performance and was selected as the representative variant for subsequent assays. Compared with wild-type KOD (KOD-WT), KOD-Sto7d demonstrated significantly improved extension efficiency that successfully amplified 7 kb targets with only 10 s elongation time, increased salt tolerance up to 120 mM NaCl for 2 kb targets, and an improved capacity to amplify long DNA fragments up to 10 kb within 4 min. In comparison with a commercially available KOD mutant fused to a dsDNA-binding protein (Sso7d from <em>Saccharolobus solfataricus</em>) at its C-terminus (KOD-Sso7d), KOD-Sto7d demonstrated greater salt tolerance and sensitivity. These results suggest that KOD-Sto7d is a robust polymerase suitable for time-saving and high-demanding PCR.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106809"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144932935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.
{"title":"Solubilization and refolding of inclusion bodies by detergents.","authors":"Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto","doi":"10.1016/j.pep.2025.106791","DOIUrl":"10.1016/j.pep.2025.106791","url":null,"abstract":"<p><p>Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106791"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-08-28DOI: 10.1016/j.pep.2025.106808
Anttoni Korkiakoski, Sami Oksanen, Tuomas Huovinen
Fed-batch fermentation results in high recombinant protein titers in limited culture volumes. Therefore, it is the preferred operation mode in the bioprocess industry. Optimizing feeding, induction, and harvest timing is a significant time-consuming challenge in bioprocessing complicated by the fact that expressed target protein is rarely detectable in real-time. In this study, the construction of an online sensor is described integrating a dual GFP promoter construct, a blue LED and a Raspberry Pi camera for real-time monitoring of recombinant antibody expression in Escherichia coli. The dual promoter construct allows simultaneous expression of GFP in the cytoplasm and the recombinant antibody in the periplasm, enabling the use of GFP fluorescence as a proxy for protein yield. GFP fluorescence correlated with Fab and nanobody expression over time and the relative quantity of fluorescence predicted the extent of induction. In nanobody fed-batch fermentations, the decreasing rate of dGFP/dt was a valuable parameter for identifying the optimal harvest point, minimizing excessive incubation time and reducing nanobody leakage into the medium. It was further demonstrated that quantitation of pixel values from RGB images captured with a Raspberry Pi 8 MP camera in the flow cell resulted in equal sensitivity for GFP detection as that achieved with a μPMT and photodiode sensors. The 3D-printable GFP sensor station is a valuable tool for process optimization and for educating bioprocess engineering students through real-time visualization of promoter activation.
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Pub Date : 2025-11-01Epub Date: 2025-07-19DOI: 10.1016/j.pep.2025.106782
Dongming Xue , Liyue Zhou , Tianxia Sun , Fang Hui , Yuming Wang , Jie Li , Ge Hui , Yu Zhao
Objective
To perform the prokaryotic expression and purification of sika deer prothymosin α (PTMA) protein and lay a foundation for the subsequent study of PTMA protein activity.
Methods
The PTMA fragment was synthesized and cloned into the pET21a-PTMA vector. Escherichia coli BL21 (DE3) was used for the prokaryotic expression of the PTMA protein, and the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nickel-containing electrophoresis. Polyacrylamide gel electrophoresis was conducted to analyze the expression products, which were purified using nickel ion affinity chromatography. Fragments were identified using mass spectrometry and analyzed for activity in MC3T3-E1 and ATDC5 cells.
Results
The pET21a-PTMA expression vector was successfully constructed, and the protein purity reached more than 93 %. The purified protein displayed proliferative activity in both MC3T3-E1 and ATDC5 cells.
Conclusion
The successful construction of a stable expression vector and production of high-purity protein lay a foundation for future research on PTMA activity and its effects on bone diseases.
{"title":"Expression, purification, and activity of sika deer prothymosin α protein","authors":"Dongming Xue , Liyue Zhou , Tianxia Sun , Fang Hui , Yuming Wang , Jie Li , Ge Hui , Yu Zhao","doi":"10.1016/j.pep.2025.106782","DOIUrl":"10.1016/j.pep.2025.106782","url":null,"abstract":"<div><h3>Objective</h3><div>To perform the prokaryotic expression and purification of sika deer prothymosin α (PTMA) protein and lay a foundation for the subsequent study of PTMA protein activity.</div></div><div><h3>Methods</h3><div>The PTMA fragment was synthesized and cloned into the pET21a-PTMA vector. <em>Escherichia coli</em> BL21 (DE3) was used for the prokaryotic expression of the PTMA protein, and the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nickel-containing electrophoresis. Polyacrylamide gel electrophoresis was conducted to analyze the expression products, which were purified using nickel ion affinity chromatography. Fragments were identified using mass spectrometry and analyzed for activity in MC3T3-E1 and ATDC5 cells.</div></div><div><h3>Results</h3><div>The pET21a-PTMA expression vector was successfully constructed, and the protein purity reached more than 93 %. The purified protein displayed proliferative activity in both MC3T3-E1 and ATDC5 cells.</div></div><div><h3>Conclusion</h3><div>The successful construction of a stable expression vector and production of high-purity protein lay a foundation for future research on PTMA activity and its effects on bone diseases.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106782"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144680570","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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