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.
{"title":"Scalable production of functional recombinant human plasma gelsolin in Escherichia coli for therapeutic and diagnostic applications","authors":"Young Su Kim , Hye-Jeong Lee , Mi-Reu Kim , Hwabong Jeong , Young Pil Kim , Jung-Ho Park , Jungoh Ahn","doi":"10.1016/j.pep.2025.106786","DOIUrl":"10.1016/j.pep.2025.106786","url":null,"abstract":"<div><div>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 <em>E. coli</em> 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 <em>E. coli</em>, 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106786"},"PeriodicalIF":1.2,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765319","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 : 2025-07-25DOI: 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":"2025-07-25","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 : 2025-07-23DOI: 10.1016/j.pep.2025.106785
Asha Kumari , Sheetal , Savitri , Monica Sharma
Hydroxynitrile lyases (HNLs) play a vital role in the asymmetric synthesis of drug precursors and plant defence. In this study, an R-specific HNL (PycHNL) was isolated and purified from Pyrus communis (pear) seeds using ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, achieving a 14.31 % yield and 6.9-fold purification. Native PAGE estimated a molecular mass of ∼92 kDa, and SDS-PAGE revealed heterodimeric subunits of 52 and 39 kDa. High-Performance Liquid Chromatography confirmed the presence of flavin adenine dinucleotide (FAD). Peptide sequencing showed no significant similarity with other Rosaceae HNLs but indicated partial identity with serine carboxypeptidases and α/β hydrolase fold proteins from Arabidopsis species. Optimal enzyme activity was observed at pH 5.5 and 30 °C, with stability for up to 6 h. Kinetic analysis revealed a Km of 11.75 mM, Vmax of 227.27 μmol/min/mg, kcat of 101.46/min, and a half-life of ∼1.9 days. Chiral HPLC analysis demonstrated that PycHNL preferentially synthesized (R)-mandelonitrile with 96.33 % enantiomeric excess and 86.83 % molar conversion, indicating its potential for biocatalytic applications in producing enantiopure nitriles.
{"title":"Novel (R)-Hydroxynitrile lyase enzyme of Pyrus communis: Purification and characterization of its physicochemical and kinetic properties","authors":"Asha Kumari , Sheetal , Savitri , Monica Sharma","doi":"10.1016/j.pep.2025.106785","DOIUrl":"10.1016/j.pep.2025.106785","url":null,"abstract":"<div><div>Hydroxynitrile lyases (HNLs) play a vital role in the asymmetric synthesis of drug precursors and plant defence. In this study, an <em>R</em>-specific HNL (PycHNL) was isolated and purified from <em>Pyrus communis</em> (pear) seeds using ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, achieving a 14.31 % yield and 6.9-fold purification. Native PAGE estimated a molecular mass of ∼92 kDa, and SDS-PAGE revealed heterodimeric subunits of 52 and 39 kDa. High-Performance Liquid Chromatography confirmed the presence of flavin adenine dinucleotide (FAD). Peptide sequencing showed no significant similarity with other Rosaceae HNLs but indicated partial identity with serine carboxypeptidases and α/β hydrolase fold proteins from <em>Arabidopsis</em> species. Optimal enzyme activity was observed at pH 5.5 and 30 °C, with stability for up to 6 h. Kinetic analysis revealed a K<sub>m</sub> of 11.75 mM, V<sub>max</sub> of 227.27 μmol/min/mg, k<sub>cat</sub> of 101.46/min, and a half-life of ∼1.9 days. Chiral HPLC analysis demonstrated that PycHNL preferentially synthesized (<em>R</em>)-mandelonitrile with 96.33 % enantiomeric excess and 86.83 % molar conversion, indicating its potential for biocatalytic applications in producing enantiopure nitriles.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106785"},"PeriodicalIF":1.2,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718340","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 : 2025-07-23DOI: 10.1016/j.pep.2025.106784
Sreejith Raran-Kurussi , Pragyan P. Parida , Akanksha Aphale , David S. Waugh
Escherichia coli remains the leading platform for recombinant protein production, yet co-expression of multiprotein complexes often suffers from stoichiometric imbalance, presenting a persistent challenge to precise subunit assembly. Many important biological functions are performed not by single proteins but by multiprotein complexes. Studying the structure and function of multiprotein complexes would be greatly facilitated if they could be reliably overproduced in heterologous host organisms. Here, we describe a simple method for the production of the human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli. Rather than producing the two chaperonin subunits from a polycistronic mRNA, a strategy commonly employed by bacterial operons, we chose to make them in the form of a single polyprotein that is subsequently cleaved inside bacterial cells by tobacco vein mottling virus (TVMV) protease. In this way, equimolar amounts of mature Hsp60 and Hsp10 subunits could be ensured. The TVMV protease is produced from a second mRNA that is transcribed from the same plasmid. Although expressed at a much lower level than the polyprotein, enough TVMV protease is produced to cleave all of the Hsp10/Hsp60 fusion protein in vivo. Moreover, we show that the mitochondrial chaperonin is fully functional when produced in this manner.
{"title":"Efficient production of human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli using a polyprotein strategy","authors":"Sreejith Raran-Kurussi , Pragyan P. Parida , Akanksha Aphale , David S. Waugh","doi":"10.1016/j.pep.2025.106784","DOIUrl":"10.1016/j.pep.2025.106784","url":null,"abstract":"<div><div><em>Escherichia coli</em> remains the leading platform for recombinant protein production, yet co-expression of multiprotein complexes often suffers from stoichiometric imbalance, presenting a persistent challenge to precise subunit assembly. Many important biological functions are performed not by single proteins but by multiprotein complexes. Studying the structure and function of multiprotein complexes would be greatly facilitated if they could be reliably overproduced in heterologous host organisms. Here, we describe a simple method for the production of the human mitochondrial chaperonin (Hsp60/Hsp10) in <em>Escherichia coli</em>. Rather than producing the two chaperonin subunits from a polycistronic mRNA, a strategy commonly employed by bacterial operons, we chose to make them in the form of a single polyprotein that is subsequently cleaved inside bacterial cells by tobacco vein mottling virus (TVMV) protease. In this way, equimolar amounts of mature Hsp60 and Hsp10 subunits could be ensured. The TVMV protease is produced from a second mRNA that is transcribed from the same plasmid. Although expressed at a much lower level than the polyprotein, enough TVMV protease is produced to cleave all of the Hsp10/Hsp60 fusion protein <em>in vivo</em>. Moreover, we show that the mitochondrial chaperonin is fully functional when produced in this manner.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106784"},"PeriodicalIF":1.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144702434","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}
Zearalenone (ZEN), a mycotoxin produced by Fusarium species, poses considerable health risks to humans and animals because of its residues in maize and its industrial byproducts. Effective methods to control or eliminate ZEN are urgently needed. Herein, Bacillus subtilis strain Z1Y3, capable of efficient ZEN transformation, was isolated from ZEN-contaminated feed samples. This strain completely transformed 5 mg/L ZEN within 15 h. Subsequently, a ZEN phosphotransferase gene, ZPH13, was identified in its genome. The gene was heterologously expressed in Escherichia coli BL21 (DE3), and the recombinant ZPH13 protein was biochemically characterised. The enzyme exhibited optimal activity at 35 °C and pH 8.0, achieving transformation of 2 μg/mL ZEN within 0.5 h under these conditions. Pythia was used to modify protein stability, identifying potential stabilization hotspots through free energy calculations (ΔΔG) and screening to obtain beneficial mutants N371I and N371T, which improved both enzyme activity and stability. Enzymatic transformation product analysis via ultra-high-performance liquid chromatography–quadrupole exactive–mass spectrometry revealed that ZEN was transformed into zearalenone-phosphate (ZEN-P) via phosphotransferase activity. Furthermore, co-culturing T-47D Human Breast Ductal Carcinoma (T47D) cells with the transformation products demonstrated a considerably reduced toxicity compared to that of untreated ZEN. These findings establish a new paradigm for mycotoxin transformation through innovative enzyme discovery and protein engineering strategies.
{"title":"Characterisation of ZPH13 phosphotransferase from Bacillus subtilis in zearalenone transformation","authors":"Yu Zhou , Feng-Jun Qiu , Ju-Bao Li , Yuan-Yuan Zhe , Zi-Xian Cheng , Qian Wu , Hua-Biao Miao , Zun-Xi Huang","doi":"10.1016/j.pep.2025.106783","DOIUrl":"10.1016/j.pep.2025.106783","url":null,"abstract":"<div><div>Zearalenone (ZEN), a mycotoxin produced by <em>Fusarium</em> species, poses considerable health risks to humans and animals because of its residues in maize and its industrial byproducts. Effective methods to control or eliminate ZEN are urgently needed. Herein, <em>Bacillus subtilis</em> strain Z1Y3, capable of efficient ZEN transformation, was isolated from ZEN-contaminated feed samples. This strain completely transformed 5 mg/L ZEN within 15 h. Subsequently, a ZEN phosphotransferase gene, <em>ZPH13</em>, was identified in its genome. The gene was heterologously expressed in <em>Escherichia coli</em> BL21 (DE3), and the recombinant ZPH13 protein was biochemically characterised. The enzyme exhibited optimal activity at 35 °C and pH 8.0, achieving transformation of 2 μg/mL ZEN within 0.5 h under these conditions. Pythia was used to modify protein stability, identifying potential stabilization hotspots through free energy calculations (ΔΔG) and screening to obtain beneficial mutants N371I and N371T, which improved both enzyme activity and stability. Enzymatic transformation product analysis via ultra-high-performance liquid chromatography–quadrupole exactive–mass spectrometry revealed that ZEN was transformed into zearalenone-phosphate (ZEN-P) via phosphotransferase activity. Furthermore, co-culturing T-47D Human Breast Ductal Carcinoma (T47D) cells with the transformation products demonstrated a considerably reduced toxicity compared to that of untreated ZEN. These findings establish a new paradigm for mycotoxin transformation through innovative enzyme discovery and protein engineering strategies.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106783"},"PeriodicalIF":1.4,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144695084","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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