Pub Date : 2025-09-22DOI: 10.1016/j.pep.2025.106822
Yong-Hong Nie , Qiang Li , Yan-Ji Lu , Tuo Tang , Xian Hong , Xin-Xin Yue , Zhi-Hui Deng , Tao Wang
C14orf119 is a functionally uncharacterized mitochondrial protein potentially implicated in ischemic stroke pathogenesis. To enable comprehensive biological studies, we developed and validated a specific polyclonal antibody against this target. Our approach involved prokaryotic expression and chromatographic purification of both His- and GST-tagged C14orf119 in Escherichia coli. The purified His-C14orf119 served as immunogen for rabbit polyclonal antibody production, while the purified GST-C14orf119 enabled subsequent affinity purification of target-specific antibody. Rigorous characterization demonstrated the antibody's exclusive specificity for both exogenous and endogenous C14orf119. Notably, the C14orf119 antibody failed to detect control His-tagged proteins, confirming effective removal of anti-His antibodies during the GST-based affinity purification process. This rigorously validated antibody provides a critical molecular tool for functional characterization of C14orf119, paving the way for mechanistic investigations into its pathophysiological roles.
{"title":"Expression and purification of C14orf119 and generation of its polyclonal antibody","authors":"Yong-Hong Nie , Qiang Li , Yan-Ji Lu , Tuo Tang , Xian Hong , Xin-Xin Yue , Zhi-Hui Deng , Tao Wang","doi":"10.1016/j.pep.2025.106822","DOIUrl":"10.1016/j.pep.2025.106822","url":null,"abstract":"<div><div>C14orf119 is a functionally uncharacterized mitochondrial protein potentially implicated in ischemic stroke pathogenesis. To enable comprehensive biological studies, we developed and validated a specific polyclonal antibody against this target. Our approach involved prokaryotic expression and chromatographic purification of both His- and GST-tagged C14orf119 in <em>Escherichia coli</em>. The purified His-C14orf119 served as immunogen for rabbit polyclonal antibody production, while the purified GST-C14orf119 enabled subsequent affinity purification of target-specific antibody. Rigorous characterization demonstrated the antibody's exclusive specificity for both exogenous and endogenous C14orf119. Notably, the C14orf119 antibody failed to detect control His-tagged proteins, confirming effective removal of anti-His antibodies during the GST-based affinity purification process. This rigorously validated antibody provides a critical molecular tool for functional characterization of C14orf119, paving the way for mechanistic investigations into its pathophysiological roles.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106822"},"PeriodicalIF":1.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145138455","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 : 2025-09-16DOI: 10.1016/j.pep.2025.106815
Jovarn V. Sullivan , Michael J. Currie , Vanessa K. Morris , Ashish Sethi , Santosh Panjikar , Grant R. Smith , Claudia-Nicole Meisrimler , Renwick C.J. Dobson
The plant disease myrtle rust is caused by the fungus Austropuccinia psidii. It has led to functional myrtaceous species extinctions in Australia and is a significant threat to other species globally. During infection, A. psidii secretes effector proteins that manipulate the host plant's defences. Numerous putative effectors are encoded in this pathogen's genome, some being expressed early during urediniospore germination and initial invasion of plant tissues. Four putative effector proteins (AP1260, AP5292, AP10948, and AP143) were found to be differentially expressed in the first 24–48 h of infection, suggesting that they play important roles in the infection process. As in other rust fungi, these effector proteins are small and cysteine-rich, often forming disulfide bonds, and their isolation for biophysical characterisation can be challenging. AlphaFold3 models predict that AP1260, AP5292, AP10948, and AP143 form disulfide bonds, while disorder analysis indicates the presence of intrinsically disordered regions. The four putative A. psidii effector proteins were recombinantly produced using SHuffle Escherichia coli cells with an adapted co-expression vector, ‘ApFunCyDisCo’. Three of the effectors were successfully produced, but were insoluble. The fourth effector, AP1260, was successfully produced in the soluble fraction and purified using a four-step process: immobilised metal affinity chromatography, desalting, anion exchange chromatography, and size exclusion chromatography. Circular dichroism spectroscopy revealed that AP1260 has a mainly random coil character, but also has both β-strand and α-helical content. This first successful production and isolation of an A. psidii protein provides a foundation for future investigation of the molecular mechanisms of A. psidii pathogenicity.
{"title":"Expression and purification of Austropuccinia psidii effector proteins in Escherichia coli","authors":"Jovarn V. Sullivan , Michael J. Currie , Vanessa K. Morris , Ashish Sethi , Santosh Panjikar , Grant R. Smith , Claudia-Nicole Meisrimler , Renwick C.J. Dobson","doi":"10.1016/j.pep.2025.106815","DOIUrl":"10.1016/j.pep.2025.106815","url":null,"abstract":"<div><div>The plant disease myrtle rust is caused by the fungus <em>Austropuccinia psidii</em>. It has led to functional myrtaceous species extinctions in Australia and is a significant threat to other species globally. During infection, <em>A. psidii</em> secretes effector proteins that manipulate the host plant's defences. Numerous putative effectors are encoded in this pathogen's genome, some being expressed early during urediniospore germination and initial invasion of plant tissues. Four putative effector proteins (AP1260, AP5292, AP10948, and AP143) were found to be differentially expressed in the first 24–48 h of infection, suggesting that they play important roles in the infection process. As in other rust fungi, these effector proteins are small and cysteine-rich, often forming disulfide bonds, and their isolation for biophysical characterisation can be challenging. AlphaFold3 models predict that AP1260, AP5292, AP10948, and AP143 form disulfide bonds, while disorder analysis indicates the presence of intrinsically disordered regions. The four putative <em>A. psidii</em> effector proteins were recombinantly produced using SHuffle <em>Escherichia coli</em> cells with an adapted co-expression vector, ‘ApFunCyDisCo’. Three of the effectors were successfully produced, but were insoluble. The fourth effector, AP1260, was successfully produced in the soluble fraction and purified using a four-step process: immobilised metal affinity chromatography, desalting, anion exchange chromatography, and size exclusion chromatography. Circular dichroism spectroscopy revealed that AP1260 has a mainly random coil character, but also has both β-strand and α-helical content. This first successful production and isolation of an <em>A. psidii</em> protein provides a foundation for future investigation of the molecular mechanisms of <em>A. psidii</em> pathogenicity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106815"},"PeriodicalIF":1.2,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086749","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}
Laccases are predominantly found in bacteria, fungi, plants, and insects, and they have numerous industrial and biotechnological applications. Laccases are utilized in the pharmaceutical, food, pulp, and paper industries, and their cost-effective production in submerged culture conditions is of significant commercial value. Attempts were made to overexpress three laccase isoforms from Trametes versicolor (TV) in heterologous systems. Recombinant TV laccases were either insoluble in E.coli or poorly expressed in Pichia pastoris. Hence a submerged fermentation process was developed to produce these commercially important laccases from two non-recombinant white rot fungi: Pleurotus ostreatus (PO) and Trametes versicolor (TV). Molasses and corn steep liquor (CSL) were used as carbon and nitrogen sources, respectively, while 2,5-xylidine, sodium lignosulfonate, and copper sulfate were used as inducers, making the entire process economical. Laccase activity reached a maximum of 374,000 U/L (or 374 kU/L) in 20 days. When evaluated at 45 °C, TV laccase outperformed PO laccase in terms of stability, a crucial factor in the delignification of biomass. TV laccase demonstrated superior stability over PO laccase at 45 °C, making it the preferred choice for biomass pre-treatment applications. We demonstrate the use of this laccase in two industrial applications.
1. Lignocellulose Depolymerization: Treatment of rice straw with Pleurotus ostreatus laccase resulted in visible structural distortion, as observed under a scanning electron microscope.
2. Industrial Wastewater Treatment: Significant decolorization of indigo carmine and Remazol Brilliant Blue dyes was achieved overnight, with reductions of up to 70 % and 74 %, respectively, when incubated with Trametes versicolor laccase.
{"title":"Optimized Laccase production from the white rot fungi Pleurotus ostreatus and Trametes versicolor","authors":"Apoorva Deshmukh , Parnal Sattikar , Aishwarya Sukhatankar , Geetanjali Wakade , Pramod Kumbhar , Phaneeswara-Rao Kommoju","doi":"10.1016/j.pep.2025.106813","DOIUrl":"10.1016/j.pep.2025.106813","url":null,"abstract":"<div><div>Laccases are predominantly found in bacteria, fungi, plants, and insects, and they have numerous industrial and biotechnological applications. Laccases are utilized in the pharmaceutical, food, pulp, and paper industries, and their cost-effective production in submerged culture conditions is of significant commercial value. Attempts were made to overexpress three laccase isoforms from <em>Trametes versicolor</em> (TV) in heterologous systems. Recombinant TV laccases were either insoluble in <em>E.coli</em> or poorly expressed in <em>Pichia pastoris.</em> Hence a submerged fermentation process was developed to produce these commercially important laccases from two non-recombinant white rot fungi: <em>Pleurotus ostreatus</em> (PO) and <em>Trametes versicolor</em> (TV). Molasses and corn steep liquor (CSL) were used as carbon and nitrogen sources, respectively, while 2,5-xylidine, sodium lignosulfonate, and copper sulfate were used as inducers, making the entire process economical. Laccase activity reached a maximum of 374,000 U/L (or 374 kU/L) in 20 days. When evaluated at 45 °C, TV laccase outperformed PO laccase in terms of stability, a crucial factor in the delignification of biomass. TV laccase demonstrated superior stability over PO laccase at 45 °C, making it the preferred choice for biomass pre-treatment applications. We demonstrate the use of this laccase in two industrial applications.</div><div>1. Lignocellulose Depolymerization: Treatment of rice straw with <em>Pleurotus ostreatus</em> laccase resulted in visible structural distortion, as observed under a scanning electron microscope.</div><div>2. Industrial Wastewater Treatment: Significant decolorization of indigo carmine and Remazol Brilliant Blue dyes was achieved overnight, with reductions of up to 70 % and 74 %, respectively, when incubated with <em>Trametes versicolor</em> laccase.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106813"},"PeriodicalIF":1.2,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058529","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 : 2025-09-11DOI: 10.1016/j.pep.2025.106814
Masafumi Katayama , Tomokazu Fukuda
Following the first report of induced pluripotent stem (iPS) cells from mouse, various mammalian-derived iPS cells have been established. In contrast, avian-derived iPS cells or iPS-like cells have been rarely reported. iPS cells can differentiate into various cell types (e.g., neural cells and hepatocytes) and proliferate indefinitely in culture. Unlike embryonic stem cells, iPS cells are generated from somatic cells, eliminating the need for embryos in their generation. Because somatic cells can be obtained from deceased individuals, iPS cell technology can be adapted for use in wild avian species, beyond its application in chickens. Our group previously reported the generation of chicken iPS cells from somatic cells using a modified-octamer-binding transcription factor 3/4 (Oct3/4), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (Klf4), MYC proto-oncogene (c-Myc), Nanog, and lin-28 homolog A (Lin28A). Developmental chicken eggs are a valuable resource for protein production. We may obtain valuable proteins from the chimeric developmental eggs of chickens using genome-edited or transgenic chicken iPS cells. Furthermore, our group established iPS cells derived from endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) using modified-Oct3/4, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2. Cells differentiated from iPS cells (e.g., neural cells and hepatocytes) can be used for drug testing in veterinary medicine and for evaluating sensitivity to infectious diseases and pollutants. We believe that iPS cell technology can be developed as a powerful tool for the conservation of endangered avian species.
{"title":"Adaptation of induced pluripotent stem cell technology for avian species","authors":"Masafumi Katayama , Tomokazu Fukuda","doi":"10.1016/j.pep.2025.106814","DOIUrl":"10.1016/j.pep.2025.106814","url":null,"abstract":"<div><div>Following the first report of induced pluripotent stem (iPS) cells from mouse, various mammalian-derived iPS cells have been established. In contrast, avian-derived iPS cells or iPS-like cells have been rarely reported. iPS cells can differentiate into various cell types (e.g., neural cells and hepatocytes) and proliferate indefinitely in culture. Unlike embryonic stem cells, iPS cells are generated from somatic cells, eliminating the need for embryos in their generation. Because somatic cells can be obtained from deceased individuals, iPS cell technology can be adapted for use in wild avian species, beyond its application in chickens. Our group previously reported the generation of chicken iPS cells from somatic cells using a modified-octamer-binding transcription factor 3/4 (Oct3/4), SRY-box transcription factor 2 (Sox2), Krüppel-like factor 4 (Klf4), MYC proto-oncogene (c-Myc), Nanog, and lin-28 homolog A (Lin28A). Developmental chicken eggs are a valuable resource for protein production. We may obtain valuable proteins from the chimeric developmental eggs of chickens using genome-edited or transgenic chicken iPS cells. Furthermore, our group established iPS cells derived from endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) using modified-Oct3/4, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2. Cells differentiated from iPS cells (e.g., neural cells and hepatocytes) can be used for drug testing in veterinary medicine and for evaluating sensitivity to infectious diseases and pollutants. We believe that iPS cell technology can be developed as a powerful tool for the conservation of endangered avian species.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106814"},"PeriodicalIF":1.2,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058524","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 : 2025-09-11DOI: 10.1016/j.pep.2025.106812
Kennosuke Ichikawa, Mike J. McGrew
Avian biology has contributed to many research areas, such as sustainable protein production, endocrinology, developmental biology, neurosciences, and immunology. Primordial germ cells, lineage-restricted stem cells, are key for the conservation of genetic diversity of bird species, as well as for studying germ cell development and producing genetic models to study avian biology. Here, we review the current knowledge of developmental and fate decision processes in avian primordial germ cells focusing on insights revealed by gene expression profiling. We summarized the characteristics and fundamental pathways required for chicken primordial cell growth. In addition, we discuss the common and disparate features of PGCs from chicken compared to other avian species. These insights are valuable for researchers in germ cell biology, reproductive biotechnology, and avian genetic conservation and indicate a need for the analysis of further bird species.
{"title":"Transcriptome profiles for defining avian primordial germ cell development","authors":"Kennosuke Ichikawa, Mike J. McGrew","doi":"10.1016/j.pep.2025.106812","DOIUrl":"10.1016/j.pep.2025.106812","url":null,"abstract":"<div><div>Avian biology has contributed to many research areas, such as sustainable protein production, endocrinology, developmental biology, neurosciences, and immunology. Primordial germ cells, lineage-restricted stem cells, are key for the conservation of genetic diversity of bird species, as well as for studying germ cell development and producing genetic models to study avian biology. Here, we review the current knowledge of developmental and fate decision processes in avian primordial germ cells focusing on insights revealed by gene expression profiling. We summarized the characteristics and fundamental pathways required for chicken primordial cell growth. In addition, we discuss the common and disparate features of PGCs from chicken compared to other avian species. These insights are valuable for researchers in germ cell biology, reproductive biotechnology, and avian genetic conservation and indicate a need for the analysis of further bird species.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"237 ","pages":"Article 106812"},"PeriodicalIF":1.2,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-05DOI: 10.1016/j.pep.2025.106811
Thanh-Hoa T. Tran , Trung-Duc Nguyen , Ngoc-Nam Phan , Hang T. Ngo , Phuc-Loc Nguyen Do , Phan-Anh Le , Nho-Thai Dinh , Tuan-Nghia Phan , Hong-Loan T. Nguyen
The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6×His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (y = 725.29x − 52.356; R2 = 0.998) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a Km of 22.01 ± 3.5 μM, kcat of 0.021 s-1, and catalytic efficiency (kcat/Km) of 946 M-1.s-1. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC50 of 0.88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.
{"title":"A magnetic bead-based fluorescent substrate for sensitive assay of SARS-CoV-2 3C-like protease activity","authors":"Thanh-Hoa T. Tran , Trung-Duc Nguyen , Ngoc-Nam Phan , Hang T. Ngo , Phuc-Loc Nguyen Do , Phan-Anh Le , Nho-Thai Dinh , Tuan-Nghia Phan , Hong-Loan T. Nguyen","doi":"10.1016/j.pep.2025.106811","DOIUrl":"10.1016/j.pep.2025.106811","url":null,"abstract":"<div><div>The 3C-like protease (3CLpro) of SARS-CoV-2 is a crucial target for antiviral drugs due to its essential role in viral polyprotein processing. In this study, we designed and produced a modular fluorescent recombinant substrate (6×His-ECFP-AVLQSGFRK-EYFP), which was then immobilized on Ni-NTA magnetic beads (Ni-NTA-6×His-ECFP-AVLQSGFRK-EYFP) for the assay of 3CLpro activity. Upon cleavage at the specific AVLQ↓SG motif, the EYFP fragment was released into the supernatant and quantified via fluorescence measurement (Ex/Em = 480/528 nm). A standard curve (<em>y</em> = <em>725.29x</em> − <em>52.356</em>; <em>R</em><sup><em>2</em></sup> = <em>0.998</em>) was obtained, enabling accurate quantification of the cleaved product and kinetic parameters. The assay using the designed substrate revealed a K<sub>m</sub> of 22.01 ± 3.5 μM, k<sub>cat</sub> of 0.021 s<sup>-</sup><sup>1</sup>, and catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub>) of 946 M<sup>-</sup><sup>1</sup><sup>.</sup>s<sup>-</sup><sup>1</sup>. The assay showed ∼50-fold greater sensitivity compared to SDS-PAGE and the inhibitory effect of GC376 for 3CLpro was also determined, with IC<sub>50</sub> of 0.88 μM. Since the modular substrate design allows for substitution of the N-terminal domain and cleavage motif, our development of the substrate and assay could be expanded to other high-specificity proteases.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106811"},"PeriodicalIF":1.2,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145016039","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-04DOI: 10.1016/j.pep.2025.106810
Hongxia Li, Xiong Wang, Xinye Wang, Zhang Zhang, Linmin Ran
Aldehyde dehydrogenase 2 (ALDH2) plays a critical role in ethanol metabolism by converting toxic acetaldehyde to acetate. To investigate its functional mechanisms and potential therapeutic applications for alcohol-related diseases, heterologous expression of ALDH2 is essential. However, ALDH2 often forms inclusion bodies when expressed in Escherichia coli. In this work, the solubility of ALDH2 was enhanced by systematic optimization of expression conditions using IPTG and lactose as respective inducers. Under optimized conditions, the media yield of ALDH2 induced by IPTG and lactose reached 44.5 ± 1.3 and 48.7 ± 1.2 μg/mL respectively, representing 7.1- and 7.8-fold improvements over unoptimized conditions. Enzymatic characterization revealed that purified ALDH2 exhibited optimal activity of 9.7 U/mL at 37 °C and pH 8.0. This research demonstrates that optimizing expression conditions is an effective strategy to enhance the solubility of recombinant enzymes, while providing a practical solution for other enzymes prone to inclusion body formation.
{"title":"High-level soluble expression of human aldehyde dehydrogenase 2 in Escherichia coli achieved through lactose-mediated induction optimization","authors":"Hongxia Li, Xiong Wang, Xinye Wang, Zhang Zhang, Linmin Ran","doi":"10.1016/j.pep.2025.106810","DOIUrl":"10.1016/j.pep.2025.106810","url":null,"abstract":"<div><div>Aldehyde dehydrogenase 2 (ALDH2) plays a critical role in ethanol metabolism by converting toxic acetaldehyde to acetate. To investigate its functional mechanisms and potential therapeutic applications for alcohol-related diseases, heterologous expression of ALDH2 is essential. However, ALDH2 often forms inclusion bodies when expressed in <em>Escherichia coli</em>. In this work, the solubility of ALDH2 was enhanced by systematic optimization of expression conditions using IPTG and lactose as respective inducers. Under optimized conditions, the media yield of ALDH2 induced by IPTG and lactose reached 44.5 ± 1.3 and 48.7 ± 1.2 μg/mL respectively, representing 7.1- and 7.8-fold improvements over unoptimized conditions. Enzymatic characterization revealed that purified ALDH2 exhibited optimal activity of 9.7 U/mL at 37 °C and pH 8.0. This research demonstrates that optimizing expression conditions is an effective strategy to enhance the solubility of recombinant enzymes, while providing a practical solution for other enzymes prone to inclusion body formation.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106810"},"PeriodicalIF":1.2,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145004120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-31DOI: 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":"2025-08-31","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}
Pub 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.
{"title":"Advancing recombinant antibody production in E. coli: Optimization of expression and purification via dual GFP promoter and imaging technology","authors":"Anttoni Korkiakoski, Sami Oksanen, Tuomas Huovinen","doi":"10.1016/j.pep.2025.106808","DOIUrl":"10.1016/j.pep.2025.106808","url":null,"abstract":"<div><div>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 <em>Escherichia coli</em>. 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.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106808"},"PeriodicalIF":1.2,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966270","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 : 2025-08-27DOI: 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":"2025-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144966316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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