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}
Pub 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":"2025-07-19","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}
Production of the receptor binding domain, RBD, variants of SARS-CoV-2, has been necessary to understand their interaction with the human receptor ACE2 and to develop alternative strategies against COVID-19. Affordable bacterial production can aid in these efforts and offer alternatives to address the ongoing emergence of new variants. Expressing recombinant RBD in E. coli could provide a valuable alternative if the challenges related to disulfide bond formation, low solubility, and the absence of glycosylation capabilities are addressed in this heterologous expression system. In this study, five representative RBD variants were expressed in E. coli BL21 (two versions of the RBD Wuhan sequence (Wt), Delta, Omicron BA.2, and Omicron JN.1). The resulting inclusion bodies were solubilized using guanidine chloride and refolded in the presence of a buffer containing the redox couple GSH-GSSG and L-arginine monohydrochloride. Purification was achieved through size-exclusion chromatography. These purified RBD variants were analyzed and compared to the RBD with the Wuhan sequence and the Omicron BA.5 variant, both produced in Chinese Hamster Ovary cells, regarding their ability to bind to ACE2 and using surface plasmon resonance, SPR, studies. The strategy outlined in this work did not include expression studies or a complete comparison of the expression yields obtained in relation to other expression systems. However, it clearly demonstrates the feasibility of obtaining various RBD variants, capable of binding to ACE2, starting from inclusion bodies, and exhibiting comparable affinities to those RBD variants produced in eukaryotic cells. This approach provides an additional option to enhance the existing tools available for research laboratories studying interactions between biologically active RBD variants and ACE2.
{"title":"Functional expression of five refolded recombinant variants of RBD from SARS-CoV-2 in Escherichia coli","authors":"Ezequiel Tobón , Lidia Riaño-Umbarila , Humberto Flores , Michelle Gutierrez , Xavier Soberón , Baltazar Becerril-Lujan , Octavio T. Ramírez , Enrique Rudiño-Piñera","doi":"10.1016/j.pep.2025.106781","DOIUrl":"10.1016/j.pep.2025.106781","url":null,"abstract":"<div><div>Production of the receptor binding domain, RBD, variants of SARS-CoV-2, has been necessary to understand their interaction with the human receptor ACE2 and to develop alternative strategies against COVID-19. Affordable bacterial production can aid in these efforts and offer alternatives to address the ongoing emergence of new variants. Expressing recombinant RBD in <em>E. coli</em> could provide a valuable alternative if the challenges related to disulfide bond formation, low solubility, and the absence of glycosylation capabilities are addressed in this heterologous expression system. In this study, five representative RBD variants were expressed in <em>E. coli</em> BL21 (two versions of the RBD Wuhan sequence (Wt), Delta, Omicron BA.2, and Omicron JN.1). The resulting inclusion bodies were solubilized using guanidine chloride and refolded in the presence of a buffer containing the redox couple GSH-GSSG and L-arginine monohydrochloride. Purification was achieved through size-exclusion chromatography. These purified RBD variants were analyzed and compared to the RBD with the Wuhan sequence and the Omicron BA.5 variant, both produced in Chinese Hamster Ovary cells, regarding their ability to bind to ACE2 and using surface plasmon resonance, SPR, studies. The strategy outlined in this work did not include expression studies or a complete comparison of the expression yields obtained in relation to other expression systems. However, it clearly demonstrates the feasibility of obtaining various RBD variants, capable of binding to ACE2, starting from inclusion bodies, and exhibiting comparable affinities to those RBD variants produced in eukaryotic cells. This approach provides an additional option to enhance the existing tools available for research laboratories studying interactions between biologically active RBD variants and ACE2.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106781"},"PeriodicalIF":1.4,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144668222","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 : 2025-07-17DOI: 10.1016/j.pep.2025.106780
Meng Li , Pan-Pan Xiao , Wan-Yu Li , Ling-Jing Zhang , Yu-Lei Chen , Tengchuan Jin , Min-Jie Cao
Angiotensin I-converting enzyme (ACE) is a key target for screening hypertension medicines. However, traditional method for ACE preparation is time-consuming, and commercial ACE is expensive. In this study, a simple and effective method for ACE purification was proposed. ACE was purified to homogeneity from porcine lungs using acid precipitation, ammonium sulfate fractionation, and chromatography on a HiTrap Q HP column. 2D-PAGE showed that the molecular weight of ACE was 180 kDa, and the pI was 5.7, its sequence was verified by LC-MS/MS. The thermal denaturation temperature of ACE was 58.8 ± 0.4 °C and it is a glycoprotein as confirmed by PAS staining. Circular dichroism and endogenous fluorescence spectroscopy showed that the addition of zinc ions led to changes in the structure of ACE, thereby inhibiting its activity. Compared with commercial ACE, the enzyme prepared in the present study exhibited higher purity and 1.3-fold higher specific activity. As ACE is widely required for studies on anti-hypertensive functional foods, effective preparation of high-purity ACE will be valuable for the investigation of potential functional foods and especially its inhibitory peptides.
{"title":"Rapid purification and characterization of angiotensin I-converting enzyme from porcine lung","authors":"Meng Li , Pan-Pan Xiao , Wan-Yu Li , Ling-Jing Zhang , Yu-Lei Chen , Tengchuan Jin , Min-Jie Cao","doi":"10.1016/j.pep.2025.106780","DOIUrl":"10.1016/j.pep.2025.106780","url":null,"abstract":"<div><div>Angiotensin I-converting enzyme (ACE) is a key target for screening hypertension medicines. However, traditional method for ACE preparation is time-consuming, and commercial ACE is expensive. In this study, a simple and effective method for ACE purification was proposed. ACE was purified to homogeneity from porcine lungs using acid precipitation, ammonium sulfate fractionation, and chromatography on a HiTrap Q HP column. 2D-PAGE showed that the molecular weight of ACE was 180 kDa, and the p<em>I</em> was 5.7, its sequence was verified by LC-MS/MS. The thermal denaturation temperature of ACE was 58.8 ± 0.4 °C and it is a glycoprotein as confirmed by PAS staining. Circular dichroism and endogenous fluorescence spectroscopy showed that the addition of zinc ions led to changes in the structure of ACE, thereby inhibiting its activity. Compared with commercial ACE, the enzyme prepared in the present study exhibited higher purity and 1.3-fold higher specific activity. As ACE is widely required for studies on anti-hypertensive functional foods, effective preparation of high-purity ACE will be valuable for the investigation of potential functional foods and especially its inhibitory peptides.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106780"},"PeriodicalIF":1.4,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144668223","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}
The glutamine synthetase (GS) enzyme pathway promotes ammonium assimilation in bacteria and is a metabolic hub for glutamine and glutamate homeostasis. Bacterial GS can be reversibly inhibited through adenylylation as a response to nitrogen availability, carried out by the GlnE enzyme. The adenylylation changes GS catalytic and regulatory properties, such as the sensitivity to negative feedback by allosteric modulators and the preferred cofactor usage. In this way, the purification of GS in different modification states can be useful during the investigation of its regulatory properties. Here we show that just by changing nitrogen availability during cell growth it is possible to obtain adenylylated or unmodified GS enzymes after heterologous expression followed by a one-step purification. As a model, we expressed GS enzymes from the diazotrophic bacteria Herbaspirillum seropedicae and Azospirillum brasilense in the M9 media supplemented with ammonium or glutamine. The enzymes were purified by Ni2+-affinity chromatography. The data showed that just by varying the nitrogen source during protein expression it was possible to obtain GS in different adenylation status. The different adenylated isoforms of GS obtained were confirmed by electrophoretic mobility shifts and showed unique responses to Mg2+ and Mn2+ ions and feedback inhibition by amino acids. Finally, we show the unmodified GS can only bind the glutamate substrate when ATP is present. The method to purify GS on different adenylylation states in a single step described here will facilitate the characterization of this key metabolic enzyme in the future.
{"title":"Modulation of Glutamine Synthetase adenylylation by nitrogen availability enables one-step purification in distinct post-translational states","authors":"Larissa Fonseca Tomazini , Eduardo Sabatine Lopes , Bárbara Barizão Nogueira , Gabriela Carvalho Calsavara , Ana Paula Santos Silva , Naiara Cristina Lucredi , Edileusa Cristina Marques Gerhardt , Luciano Fernandes Huergo , Marco Aurelio Schuler Oliveira","doi":"10.1016/j.pep.2025.106779","DOIUrl":"10.1016/j.pep.2025.106779","url":null,"abstract":"<div><div>The glutamine synthetase (GS) enzyme pathway promotes ammonium assimilation in bacteria and is a metabolic hub for glutamine and glutamate homeostasis. Bacterial GS can be reversibly inhibited through adenylylation as a response to nitrogen availability, carried out by the GlnE enzyme. The adenylylation changes GS catalytic and regulatory properties, such as the sensitivity to negative feedback by allosteric modulators and the preferred cofactor usage. In this way, the purification of GS in different modification states can be useful during the investigation of its regulatory properties. Here we show that just by changing nitrogen availability during cell growth it is possible to obtain adenylylated or unmodified GS enzymes after heterologous expression followed by a one-step purification. As a model, we expressed GS enzymes from the diazotrophic bacteria <em>Herbaspirillum seropedicae</em> and <em>Azospirillum brasilense</em> in the M9 media supplemented with ammonium or glutamine. The enzymes were purified by Ni<sup>2+</sup>-affinity chromatography. The data showed that just by varying the nitrogen source during protein expression it was possible to obtain GS in different adenylation status. The different adenylated isoforms of GS obtained were confirmed by electrophoretic mobility shifts and showed unique responses to Mg<sup>2+</sup> and Mn<sup>2+</sup> ions and feedback inhibition by amino acids. Finally, we show the unmodified GS can only bind the glutamate substrate when ATP is present. The method to purify GS on different adenylylation states in a single step described here will facilitate the characterization of this key metabolic enzyme in the future.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106779"},"PeriodicalIF":1.4,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144655550","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 : 2025-07-08DOI: 10.1016/j.pep.2025.106768
James Liu , Tongyi Dou , Jiansen Jiang , Yi He , Robert B. Hufnagel
PNPLA6 encodes neuropathy target esterase (NTE), a membrane-associated protein located in the endoplasmic reticulum and highly expressed in the developing and adult human brain and eye. Although research has uncovered the biochemical and cellular roles NTE plays in the cell, it is still unclear how NTE enzymatic activity causes defects in affected tissue, and how the structure of the protein modulates enzyme activity. Furthermore, full-length NTE has yet to be purified, which would be essential for future therapies such as enzyme replacement therapy. To address this, we developed a procedure to robustly express human full-length NTE in human suspension cell culture. This procedure expressed wild type NTE, variant PNPLA6 constructs, and N- and C-terminal tagged full-length constructs. Extraction and purification of NTE from native membranes was accomplished by synthetic nanodiscs, with CyclAPol C8-C0-50 able to solubilize and retain the activity of the full-length protein. This purified product produced homogenous populations of NTE under electron microscopy by negative staining. Overall, we provide a blueprint for large-scale expression and purification of a membrane-associated protein that is critical for studying NTE's structure and function in human disease, including therapeutic applications.
{"title":"A direct solubilization approach to purify active full-length human neuropathy target esterase","authors":"James Liu , Tongyi Dou , Jiansen Jiang , Yi He , Robert B. Hufnagel","doi":"10.1016/j.pep.2025.106768","DOIUrl":"10.1016/j.pep.2025.106768","url":null,"abstract":"<div><div><em>PNPLA6</em> encodes neuropathy target esterase (NTE), a membrane-associated protein located in the endoplasmic reticulum and highly expressed in the developing and adult human brain and eye. Although research has uncovered the biochemical and cellular roles NTE plays in the cell, it is still unclear how NTE enzymatic activity causes defects in affected tissue, and how the structure of the protein modulates enzyme activity. Furthermore, full-length NTE has yet to be purified, which would be essential for future therapies such as enzyme replacement therapy. To address this, we developed a procedure to robustly express human full-length NTE in human suspension cell culture. This procedure expressed wild type NTE, variant <em>PNPLA6</em> constructs, and N- and C-terminal tagged full-length constructs. Extraction and purification of NTE from native membranes was accomplished by synthetic nanodiscs, with CyclAPol C8-C0-50 able to solubilize and retain the activity of the full-length protein. This purified product produced homogenous populations of NTE under electron microscopy by negative staining. Overall, we provide a blueprint for large-scale expression and purification of a membrane-associated protein that is critical for studying NTE's structure and function in human disease, including therapeutic applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106768"},"PeriodicalIF":1.4,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144596635","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 : 2025-07-03DOI: 10.1016/j.pep.2025.106767
Luis Giovani de Oliveira Guardalini , Thaissa Consoni Bernardino , Felipe Moura Dias , Jaci Leme , Henrique Coelho de Oliveira , Aldo Tonso , Eutimio Gustavo Fernández Núñez , Soraia Attie Calil Jorge
The COVID-19 pandemic has led to a race to find prevention methods, such as vaccines. Virus-like particles (VLP), mimicking native viruses without genetic material, represent a promising vaccine platform due to their safety and strong immunogenicity. The baculovirus expression system, using insect cells like Sf9, offers an efficient method for VLP production. This study investigated the coinfection of Sf9 cells with recombinant baculoviruses carrying genes for SARS-CoV-2 spike, membrane, nucleocapsid, and envelope proteins in stirred tank bioreactors. Spike protein expression, VLP formation, and cellular metabolic profiles were analyzed. Bioreactor assays revealed suitable conditions for VLP production, demonstrating the robustness of this platform to handle emerging viral variants. These findings support the large-scale production of VLP carrying spike protein as the main immunogen as a versatile platform for vaccine development against SARS-CoV-2 and other coronaviruses and future viral threats, enabling a rapid response to emerging outbreaks with few changes.
{"title":"Scalable approach for coronavirus-like particles making based on the spike protein using Sf9 cells","authors":"Luis Giovani de Oliveira Guardalini , Thaissa Consoni Bernardino , Felipe Moura Dias , Jaci Leme , Henrique Coelho de Oliveira , Aldo Tonso , Eutimio Gustavo Fernández Núñez , Soraia Attie Calil Jorge","doi":"10.1016/j.pep.2025.106767","DOIUrl":"10.1016/j.pep.2025.106767","url":null,"abstract":"<div><div>The COVID-19 pandemic has led to a race to find prevention methods, such as vaccines. Virus-like particles (VLP), mimicking native viruses without genetic material, represent a promising vaccine platform due to their safety and strong immunogenicity. The baculovirus expression system, using insect cells like Sf9, offers an efficient method for VLP production. This study investigated the coinfection of Sf9 cells with recombinant baculoviruses carrying genes for SARS-CoV-2 spike, membrane, nucleocapsid, and envelope proteins in stirred tank bioreactors. Spike protein expression, VLP formation, and cellular metabolic profiles were analyzed. Bioreactor assays revealed suitable conditions for VLP production, demonstrating the robustness of this platform to handle emerging viral variants. These findings support the large-scale production of VLP carrying spike protein as the main immunogen as a versatile platform for vaccine development against SARS-CoV-2 and other coronaviruses and future viral threats, enabling a rapid response to emerging outbreaks with few changes.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106767"},"PeriodicalIF":1.4,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144567752","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 : 2025-06-28DOI: 10.1016/j.pep.2025.106765
Nadia Hadiji-Abbes, Nihel Ammous-Boukhris, Istabrak Borchani-Chabchoub, Ali Gargouri, Raja Mokdad-Gargouri
Recombinant hepatitis B surface antigen (PreS2-S) was produced in the yeast Saccharomyces cerevisiae and evaluated as a vaccine candidate against Hepatitis B Virus (HBV) infection. Recombinant virus-like particles (VLPs) were the first VLP-based vaccines to be approved. Our previous studies demonstrated high intracellular production of recombinant HBsAg using Pichia pastoris and Saccharomyces cerevisiae, along with optimized VLP extraction methods. In the present study, we aim to simplify the purification and characterization of recombinant M protein (PreS2 + S). To achieve this, we employed a two-step purification process, beginning with size exclusion chromatography followed by ion exchange chromatography that has the advantage of effectively separate the medium and large particles. The purified recombinant M protein successfully revealed the presence of VLPs, which are essential for antigenicity.
{"title":"Purification of the HBV middle protein by two simple steps, application of ion exchange chromatography to improve protein homogeneity","authors":"Nadia Hadiji-Abbes, Nihel Ammous-Boukhris, Istabrak Borchani-Chabchoub, Ali Gargouri, Raja Mokdad-Gargouri","doi":"10.1016/j.pep.2025.106765","DOIUrl":"10.1016/j.pep.2025.106765","url":null,"abstract":"<div><div>Recombinant hepatitis B surface antigen (PreS2-S) was produced in the yeast <em>Saccharomyces cerevisiae</em> and evaluated as a vaccine candidate against Hepatitis B Virus (HBV) infection. Recombinant virus-like particles (VLPs) were the first VLP-based vaccines to be approved. Our previous studies demonstrated high intracellular production of recombinant HBsAg using <em>Pichia pastoris</em> and <em>Saccharomyces cerevisiae</em>, along with optimized VLP extraction methods. In the present study, we aim to simplify the purification and characterization of recombinant M protein (PreS2 + S). To achieve this, we employed a two-step purification process, beginning with size exclusion chromatography followed by ion exchange chromatography that has the advantage of effectively separate the medium and large particles. The purified recombinant M protein successfully revealed the presence of VLPs, which are essential for antigenicity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"234 ","pages":"Article 106765"},"PeriodicalIF":1.4,"publicationDate":"2025-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144529412","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}
The HIV-1 capsid protein (CA) is essential for viral replication and serves as a validated antiviral drug target. Traditional purification of CA relies on multi-step chromatographic protocols, which are time-consuming and labor-intensive. In this study, we established a rapid, column-free purification strategy using a cleavable self-aggregating tag (cSAT) to produce functional wild-type CA protein from E. coli with >95 % purity within a single day. The workflow is compatible with high-throughput formats and scalable from microplates to fermenters, offering significant advantages over conventional purification methods. The purified CA retained full biological activity, as demonstrated by its ability to assemble into higher-order structures in a salt- and protein concentration–dependent manner in vitro. We further evaluated the effects of two well-characterized capsid modulators: CAI, a peptide inhibitor, and lenacapavir (LEN), a clinically approved capsid-targeting drug. Turbidity-based assembly assays confirmed that CAI inhibited and LEN enhanced CA assembly in a dose-dependent manner. When co-administered, CAI and LEN exhibited mutually antagonistic effects. Preincubation with CAI abolished LEN-mediated enhancement, indicating a potential conformational lock imposed by CAI. These findings demonstrate that the column-free strategy enables efficient production of functionally active CA protein suitable for downstream biochemical and inhibitor screening assays. The approach provides a practical tool for accelerating HIV-1 capsid research and antiviral discovery.
{"title":"Column-free purification of functional HIV-1 capsid protein and its application in assembly and inhibitor assays","authors":"Da-Wei Zhang , Xiao-Shuang Xu , Yimin Li , Shan Chang","doi":"10.1016/j.pep.2025.106766","DOIUrl":"10.1016/j.pep.2025.106766","url":null,"abstract":"<div><div>The HIV-1 capsid protein (CA) is essential for viral replication and serves as a validated antiviral drug target. Traditional purification of CA relies on multi-step chromatographic protocols, which are time-consuming and labor-intensive. In this study, we established a rapid, column-free purification strategy using a cleavable self-aggregating tag (cSAT) to produce functional wild-type CA protein from E. coli with >95 % purity within a single day. The workflow is compatible with high-throughput formats and scalable from microplates to fermenters, offering significant advantages over conventional purification methods. The purified CA retained full biological activity, as demonstrated by its ability to assemble into higher-order structures in a salt- and protein concentration–dependent manner in vitro. We further evaluated the effects of two well-characterized capsid modulators: CAI, a peptide inhibitor, and lenacapavir (LEN), a clinically approved capsid-targeting drug. Turbidity-based assembly assays confirmed that CAI inhibited and LEN enhanced CA assembly in a dose-dependent manner. When co-administered, CAI and LEN exhibited mutually antagonistic effects. Preincubation with CAI abolished LEN-mediated enhancement, indicating a potential conformational lock imposed by CAI. These findings demonstrate that the column-free strategy enables efficient production of functionally active CA protein suitable for downstream biochemical and inhibitor screening assays. The approach provides a practical tool for accelerating HIV-1 capsid research and antiviral discovery.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"234 ","pages":"Article 106766"},"PeriodicalIF":1.4,"publicationDate":"2025-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144517787","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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