Protein expression and purification最新文献_第8页

Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation 人Gb3/CD77合成酶在昆虫和人细胞中的表达：活性和糖基化的比较

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-18 DOI: 10.1016/j.pep.2025.106803

Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski

{"title":"Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation","authors":"Krzysztof Mikolajczyk , Katarzyna Szymczak-Kulus , Anna Bereznicka , Radoslaw Kaczmarek , Lukasz Filip Sobala , Anna Jakubiak-Augustyn , Marcin Czerwinski","doi":"10.1016/j.pep.2025.106803","DOIUrl":"10.1016/j.pep.2025.106803","url":null,"abstract":"<div><div>Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (α1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: <em>Trichoplusia ni</em> insect cells (High Five) and human embryonic kidney cells (Expi293F™). The High Five cells generate short oligomannose structures, while the Expi293F™ cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F™-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F™ cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F™-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106803"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144912957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P 未折叠蛋白反应激活因子基因HAC1P的共表达促进毕赤酵母茎菠萝蛋白酶的产生

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-20 DOI: 10.1016/j.pep.2025.106804

Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan

{"title":"Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P","authors":"Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan","doi":"10.1016/j.pep.2025.106804","DOIUrl":"10.1016/j.pep.2025.106804","url":null,"abstract":"<div><div>Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant <em>Ananas comosus</em>. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) <em>Hac1</em> on folding and secretion of recombinant stem bromelain in <em>Pichia pastoris.</em> Stem bromelain gene (BL) from <em>Ananas comosus</em> was codon optimized and expressed in the <em>Pichia pastoris</em> X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, <em>Hac1p</em> was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when <em>HAC1</em> was co-expressed with stem bromelain (BL). Fed batch studies were performed for both <em>pGAPαBL1</em> and <em>pGAPBLHAC1</em> clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones <em>like Kar2p, Sec 63, Pdi, Cne1</em>. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies <em>Pichia pastoris</em> as a robust platform to produce stem bromelain for various industrial applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106804"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144918027","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d 通过融合tokodaisulfolobus Sto7d提高柯达热球菌KOD1聚合酶的PCR性能

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-31 DOI: 10.1016/j.pep.2025.106809

Leheng Chen , Dawei Fu

{"title":"Enhanced performance of Thermococcus kodakarensis KOD1 polymerase in PCR via fusion to Sulfolobus tokodaii Sto7d","authors":"Leheng Chen , Dawei Fu","doi":"10.1016/j.pep.2025.106809","DOIUrl":"10.1016/j.pep.2025.106809","url":null,"abstract":"<div><div>The DNA polymerase from <em>Thermococcus kodakarensis</em> KOD1 (KOD) is widely utilized in polymerase chain reaction (PCR) due to its high processivity and fidelity. However, like many other B-family DNA polymerases, it faces limitations in extension efficiency, amplicon length, and resistance to PCR inhibitors. In order to further enhance its capability, novel mutants were engineered by fusing a 7 kDa nonspecific double-stranded DNA (dsDNA)-binding protein from <em>Sulfolobus tokodaii</em> (Sto7d) to the C-terminus of KOD via distinct peptide linkers, resulting in a set of KOD-Sto7d polymerase variants. These constructs were expressed, purified, and characterized. Among the variants, KOD-GT4G-Sto7d exhibited the best PCR performance and was selected as the representative variant for subsequent assays. Compared with wild-type KOD (KOD-WT), KOD-Sto7d demonstrated significantly improved extension efficiency that successfully amplified 7 kb targets with only 10 s elongation time, increased salt tolerance up to 120 mM NaCl for 2 kb targets, and an improved capacity to amplify long DNA fragments up to 10 kb within 4 min. In comparison with a commercially available KOD mutant fused to a dsDNA-binding protein (Sso7d from <em>Saccharolobus solfataricus</em>) at its C-terminus (KOD-Sso7d), KOD-Sto7d demonstrated greater salt tolerance and sensitivity. These results suggest that KOD-Sto7d is a robust polymerase suitable for time-saving and high-demanding PCR.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"236 ","pages":"Article 106809"},"PeriodicalIF":1.2,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144932935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Discovery and development of bispecific antibodies. 双特异性抗体的发现和发展。

IF 1.8 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-05 DOI: 10.1016/j.pep.2025.106787

Tetsuya Wakabayashi, Taichi Kuramochi

{"title":"Discovery and development of bispecific antibodies.","authors":"Tetsuya Wakabayashi, Taichi Kuramochi","doi":"10.1016/j.pep.2025.106787","DOIUrl":"10.1016/j.pep.2025.106787","url":null,"abstract":"<p><p>Bispecific antibodies (BsAbs) represent a significant breakthrough in antibody-based therapeutics, offering the unique capability to engage two distinct targets simultaneously. BsAbs are expected to exert therapeutic effects that are unattainable with conventional antibody drugs. Specifically, they are being developed for use in intercellular bridging, proximity effects, dual target inhibition, and cell targeting dependent on two antigen types. In recent years, antibody drug discovery has made progress by taking advantage of this dual-targeting ability, and bispecific antibodies have been launched across multiple therapeutic areas. These include antitumor drugs intended to enhance T-cell killing activity and inhibit growth factors, drugs that mimic blood coagulation factor functions, and angiogenesis inhibitors. This review highlights the pivotal technological advancements that have overcome the manufacturing challenges associated with BsAbs, enabling the development of pharmaceutical-grade products. We use emicizumab as a case study to illustrate these developments. Particular emphasis is placed on the critical synergy between antibody engineering technology and protein purification technologies, which has played a crucial role in the successful production of BsAbs. Furthermore, we discuss recent innovations in affinity chromatography, specifically the development of alkaline-resistant Protein L resins that have significantly improved commercial production processes. We examine the unique affinity behaviors of these resins and their impact on BsAb purification. This comprehensive review aims to provide researchers and industry professionals with a thorough understanding of the current landscape and future potential of bispecific antibodies in therapeutic applications, highlighting both technical challenges and innovative solutions in this rapidly evolving field.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106787"},"PeriodicalIF":1.8,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144776016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Advancing recombinant antibody production in E. coli: Optimization of expression and purification via dual GFP promoter and imaging technology 推进重组抗体在大肠杆菌中的生产：利用双GFP启动子和成像技术优化表达和纯化。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-28 DOI: 10.1016/j.pep.2025.106808

Anttoni Korkiakoski, Sami Oksanen, Tuomas Huovinen

{"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":"2025-12-01","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}

引用次数: 0

Solubilization and refolding of inclusion bodies by detergents. 洗涤剂对包裹体的增溶和再折叠作用。

IF 1.8 4区生物学

Protein expression and purification Pub Date : 2025-12-01 Epub Date: 2025-08-07 DOI: 10.1016/j.pep.2025.106791

Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto

{"title":"Solubilization and refolding of inclusion bodies by detergents.","authors":"Tsutomu Arakawa, Teruo Akuta, Daisuke Ejima, Kouhei Tsumoto","doi":"10.1016/j.pep.2025.106791","DOIUrl":"10.1016/j.pep.2025.106791","url":null,"abstract":"<p><p>Recombinant proteins play many important roles in development of biological reagents and biopharmaceuticals. Here, we will mainly review refolding of recombinant proteins when expressed in inclusion bodies, although strategies to enhance soluble expression are described as an alternative to refolding inclusion bodies. These strategies include, but not limited to, adding chemical chaperones in cell culture media, modifying cell lysis buffer and using solubility-enhacing fusion tags. Another solubility enhancement was to generate lipid complex for membrane proteins that form insoluble proteins without lipid. Among various solubilization and refolding technologies, those using denaturant, alkaline pH and pressure are also desribed, while we focus on solubilization and refolding using detergents, which are effective and cost-friendly. Sodium dodecylsulfate, lauroyl-glutamate, sarkosyl and cetyltrimethylammonium have been extensively used, as summarized in this review. Slow or step-wise removal of denaturants or ionic detergents used to solubilize appears to play a critical role in successful refolding by maintaining the solubility of proteins during refolding. In alkaline refolding, slow pH adjustment also helps maintain protein solubility. In pressure refolding, small amount of guanidine hydrochloride assisted refolding.</p>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":" ","pages":"106791"},"PeriodicalIF":1.8,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expression, purification, and activity of sika deer prothymosin α protein 梅花鹿胸腺蛋白酶α原蛋白的表达、纯化及活性研究

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-19 DOI: 10.1016/j.pep.2025.106782

Dongming Xue , Liyue Zhou , Tianxia Sun , Fang Hui , Yuming Wang , Jie Li , Ge Hui , Yu Zhao

{"title":"Expression, purification, and activity of sika deer prothymosin α protein","authors":"Dongming Xue , Liyue Zhou , Tianxia Sun , Fang Hui , Yuming Wang , Jie Li , Ge Hui , Yu Zhao","doi":"10.1016/j.pep.2025.106782","DOIUrl":"10.1016/j.pep.2025.106782","url":null,"abstract":"<div><h3>Objective</h3><div>To perform the prokaryotic expression and purification of sika deer prothymosin α (PTMA) protein and lay a foundation for the subsequent study of PTMA protein activity.</div></div><div><h3>Methods</h3><div>The PTMA fragment was synthesized and cloned into the pET21a-PTMA vector. <em>Escherichia coli</em> BL21 (DE3) was used for the prokaryotic expression of the PTMA protein, and the expression products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nickel-containing electrophoresis. Polyacrylamide gel electrophoresis was conducted to analyze the expression products, which were purified using nickel ion affinity chromatography. Fragments were identified using mass spectrometry and analyzed for activity in MC3T3-E1 and ATDC5 cells.</div></div><div><h3>Results</h3><div>The pET21a-PTMA expression vector was successfully constructed, and the protein purity reached more than 93 %. The purified protein displayed proliferative activity in both MC3T3-E1 and ATDC5 cells.</div></div><div><h3>Conclusion</h3><div>The successful construction of a stable expression vector and production of high-purity protein lay a foundation for future research on PTMA activity and its effects on bone diseases.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"235 ","pages":"Article 106782"},"PeriodicalIF":1.4,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144680570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A direct solubilization approach to purify active full-length human neuropathy target esterase 直接增溶法纯化活性全长人神经病变靶酯酶

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-08 DOI: 10.1016/j.pep.2025.106768

James Liu , Tongyi Dou , Jiansen Jiang , Yi He , Robert B. Hufnagel

{"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":"2025-11-01","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}

引用次数: 0

Novel (R)-Hydroxynitrile lyase enzyme of Pyrus communis: Purification and characterization of its physicochemical and kinetic properties 新型梨(R)-羟基腈裂解酶的纯化及其理化和动力学性质的表征。

IF 1.2 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-23 DOI: 10.1016/j.pep.2025.106785

Asha Kumari , Sheetal , Savitri , Monica Sharma

{"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":"2025-11-01","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}

引用次数: 0

Efficient production of human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli using a polyprotein strategy 利用多蛋白策略在大肠杆菌中高效生产人线粒体伴侣蛋白(Hsp60/Hsp10

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-11-01 Epub Date: 2025-07-23 DOI: 10.1016/j.pep.2025.106784

Sreejith Raran-Kurussi , Pragyan P. Parida , Akanksha Aphale , David S. Waugh

{"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":"2025-11-01","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}

引用次数: 0