Protein expression and purification最新文献_第4页

A systematic approach for scalable purification of virus-like particles 一种可扩展的病毒样颗粒纯化的系统方法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-17 DOI: 10.1016/j.pep.2025.106664

Enoch Y. Park , Robert Minkner

{"title":"A systematic approach for scalable purification of virus-like particles","authors":"Enoch Y. Park , Robert Minkner","doi":"10.1016/j.pep.2025.106664","DOIUrl":"10.1016/j.pep.2025.106664","url":null,"abstract":"<div><div>Virus-like particles (VLPs) are increasingly recognized as promising vaccine candidates and drug-delivery platforms because they do not contain genetic materials, mimic viral structures, and possess strong antigenic properties. Various hosts, including microorganisms, yeast, and insect cells, are commonly used for VLP expression. Recently, silkworms have emerged as a significant host for producing VLPs, providing a cost-effective and straightforward approach for large-scale expression. Despite the progress in VLP expression technology, purification methods for VLPs are still in their infancy and often rely on unscalable ultracentrifugation techniques. Moreover, VLP purification represents a substantial portion of the overall production cost, highlighting the urgent need for efficient and scalable downstream processing methods to overcome the current challenges in VLP production. Considering their differing structures and properties, this review systematically summarizes the published results of scalable downstream processes for both enveloped and non-enveloped VLPs. Its aim is to provide a comprehensive overview and significantly contribute to developing future VLP production for pharmaceutical applications, thereby guiding and inspiring further research in this field.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106664"},"PeriodicalIF":1.4,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143010350","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

Advantage of leaky expression, acid solubilization and CHAPS in the production of cost-effective bone morphogenetic Protein-2 漏表达、酸溶解和CHAPS在高效骨形态发生蛋白-2生产中的优势。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-11 DOI: 10.1016/j.pep.2025.106662

Nitika Patwa, Hirah Amir, Shashank Deep

引用次数: 0

Optimized isolation of enzymatically active ubiquitin E3 ligase E6AP/UBE3A from mammalian cells 优化了泛素E3连接酶E6AP/UBE3A的哺乳动物表达体系。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-09 DOI: 10.1016/j.pep.2025.106661

Johanna M. Schafer , Christine S. Muli , Rehab A. Heikal , Marzena A. Dyba , Sergey G. Tarasov , Margaret M. Stratton , Eric R. Strieter , Kylie J. Walters

{"title":"Optimized isolation of enzymatically active ubiquitin E3 ligase E6AP/UBE3A from mammalian cells","authors":"Johanna M. Schafer , Christine S. Muli , Rehab A. Heikal , Marzena A. Dyba , Sergey G. Tarasov , Margaret M. Stratton , Eric R. Strieter , Kylie J. Walters","doi":"10.1016/j.pep.2025.106661","DOIUrl":"10.1016/j.pep.2025.106661","url":null,"abstract":"<div><div>E6AP/UBE3A is the founding member of the HECT (<u>H</u>omologous to the <u>E</u>6-AP <u>C</u>arboxyl <u>T</u>erminus) ubiquitin E3 ligase family, which add ubiquitin post-translationally to protein substrates. E6AP has been structurally defined in complex with human papillomavirus (HPV) oncoprotein E6 and its gain-of-function substrate tumor suppressor p53; however, there is currently no report of E6AP being expressed and purified from mammalian cells, as studies to date have isolated E6AP from <em>E. coli</em> or insect cells. Here, we report an optimized protocol for purifying E6AP from suspended Human Embryonic Kidney (HEK) cells. Biophysical characterization by Q-TOF confirmed sample purity while mass photometry indicated that purified E6AP forms a monomer-oligomer mixture. E6AP produced by this method is catalytically active and amenable to structural characterization by cryo-electron microscopy (cryo-EM), biochemical assays, and small molecule screening campaigns.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106661"},"PeriodicalIF":1.4,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972092","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 and purification of Mycobacterium tuberculosis F420-dependent glucose-6-phosphate dehydrogenase enzyme using Escherichia coli 利用大肠杆菌表达和纯化结核分枝杆菌f420依赖性葡萄糖-6-磷酸脱氢酶。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-06 DOI: 10.1016/j.pep.2024.106650

Adewale Victor Aderemi , Matthew Snee , Richard B. Tunnicliffe , Marina Golovanova , Kathleen M. Cain , Andrew W. Munro , Jonathan P. Waltho , David Leys

{"title":"Expression and purification of Mycobacterium tuberculosis F420-dependent glucose-6-phosphate dehydrogenase enzyme using Escherichia coli","authors":"Adewale Victor Aderemi , Matthew Snee , Richard B. Tunnicliffe , Marina Golovanova , Kathleen M. Cain , Andrew W. Munro , Jonathan P. Waltho , David Leys","doi":"10.1016/j.pep.2024.106650","DOIUrl":"10.1016/j.pep.2024.106650","url":null,"abstract":"<div><div>Since their discovery in <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>), F<sub>420</sub>-dependent enzymes have been identified as both important drug targets and potential industrial biocatalysts, including for bioremediation of otherwise recalcitrant substrates. <em>Mtb</em>-FGD1, utilizes glucose 6-phosphate (G6P) as an electron donor for the reduction of F<sub>420</sub>. Current expression systems for <em>Mtb</em>-FGD1 use <em>Mycobacterium smegmatis</em> as host, because of the tendency for it to form inclusion bodies in <em>E. coli</em>. However, large scale recombinant protein production using <em>M. smegmatis</em> is slow and costly and the organism is not generally recognized as safe. Here, we report a faster, cheaper and safer approach for the expression of fully functional <em>Mtb</em>-FGD1 in <em>E. coli</em> using cold-adapted GroEL/ES as chaperones. Our approach yielded ∼70 mg of protein per litre (L) of culture. The purified enzyme catalysed the reduction of F<sub>420</sub> to F<sub>420</sub>.H<sub>2</sub> in the presence of G6P, and the re-oxidation of the F<sub>420</sub>.H<sub>2</sub> to F<sub>420</sub> when coupled to <em>Tfu</em>-FNO, which is a thermostable oxidoreductase that utilizes F<sub>420</sub> for the reversible oxidation of NADPH. This latter finding provides opportunity for the utilization of <em>Mtb</em>-FGD1 as an industrial biocatalyst or in the detoxification of environmental contaminants such as malachite green, picrate and aflatoxin.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106650"},"PeriodicalIF":1.4,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142953985","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 and purification of an activated orexin receptor 1- G-protein complex 活化的食欲素受体1- g蛋白复合物的表达和纯化。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-04 DOI: 10.1016/j.pep.2025.106660

Ioanna Ramou , Steven Janvier , Sarah Druwé , Charanne Sys , Lies Dekeyzer , Pieter Claes , Els Pardon , Christel Menet , Jan Steyaert

{"title":"Expression and purification of an activated orexin receptor 1- G-protein complex","authors":"Ioanna Ramou , Steven Janvier , Sarah Druwé , Charanne Sys , Lies Dekeyzer , Pieter Claes , Els Pardon , Christel Menet , Jan Steyaert","doi":"10.1016/j.pep.2025.106660","DOIUrl":"10.1016/j.pep.2025.106660","url":null,"abstract":"<div><div>Orexin receptors constitute a family of class A G-protein coupled receptors. There are two subtypes of orexin receptors, namely OX1R and OX2R. OX1R and OX2R are widely distributed in the central nervous system and are the targets for the peptide neurotransmitters orexin-A and orexin-B. Orexins are involved in a plethora of key physiological functions such as regulation of the sleep/wake cycle, feeding behavior, energy homeostasis, and cognition. Dysfunction of the orexin system has been linked to various pathological conditions, such as narcolepsy, insomnia, obesity, addiction, cognitive impairment, and depression. The active state structure of OX2R has been elucidated, while the active state structure of OX1R remains unresolved. Here, we describe a method for the expression and purification of an activated OX1R bound to its native peptide ligand, orexin-A, in complex with a Dominant Negative Gsq protein and Nb35. The proteins were expressed in Hi5 insect cells and subsequently purified via two consecutive affinity chromatography steps, followed by a final polishing Size Exclusion Chromatography step. This study could stimulate further research into the activation mechanisms of OX1R and the structural determination of its active state structure.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106660"},"PeriodicalIF":1.4,"publicationDate":"2025-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142953986","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

Development and selection of candidate monoclonal antibodies for the detection of Clostridium botulinum neurotoxin serotype B light chain B型轻链肉毒梭菌神经毒素候选单克隆抗体的制备及筛选。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-02 DOI: 10.1016/j.pep.2025.106659

Toan Van Trinh , Doai Van Nguyen , Hieu Dang Hoang , Hung Viet Pham , Duong Ngoc Vu , Phan Van Le , Diep Ngoc Le , Cuong Viet Vo , Lan Anh Thi Le

{"title":"Development and selection of candidate monoclonal antibodies for the detection of Clostridium botulinum neurotoxin serotype B light chain","authors":"Toan Van Trinh , Doai Van Nguyen , Hieu Dang Hoang , Hung Viet Pham , Duong Ngoc Vu , Phan Van Le , Diep Ngoc Le , Cuong Viet Vo , Lan Anh Thi Le","doi":"10.1016/j.pep.2025.106659","DOIUrl":"10.1016/j.pep.2025.106659","url":null,"abstract":"<div><div>Botulinum neurotoxin, produced by the bacterium <em>Clostridium botulinum</em>, causes botulism, a severe, rapidly progressing, and potentially fatal condition. Swift detection of the toxin and timely administration of antitoxin antibodies are critical for effective treatment. The current standard for Botulinum toxin testing is the mouse lethality assay, but this method is time-consuming and requires live animals. Consequently, a key focus of research is the development of antibodies for both diagnostic purposes and toxin neutralization. Botulinum neurotoxin serotype B (BoNT/B), one of the most dangerous and prevalent serotypes, is commonly involved in poisoning cases. Like other botulinum toxins, BoNT/B consists of heavy and light chains. In this study, we generated mouse monoclonal antibodies targeting the BoNT/B light chain (BoNT/B-LC) through hybridoma cell line development. Two monoclonal hybridomas (3B7 and 3C6) were selected from a pool of 18 polyclonal hybridomas and used to produce anti-BoNT/B-LC antibodies through the ascites fluid production. The antibodies were utilized for indirect ELISA detection of recombinant BoNT/B-LC. Notably, the assay with 3B7 demonstrated higher sensitivity, allowing for the detection of TrxA-fused BoNT/B-LC (68.9 kDa) at concentrations as low as 4 ng/mL. These results highlight the potential of the generated antibodies for rapid BoNT/B detection, offering a promising alternative to animal-based testing.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106659"},"PeriodicalIF":1.4,"publicationDate":"2025-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142927876","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

Isolation, expression, and characterization of potato (Solanum tuberosum) GH family 17 β-1,3-glucanase (Stglu) for exploring its potential as an antifungal agent 马铃薯（Solanum tuberosum） GH家族17 β-1,3-葡聚糖酶（Stglu）的分离、表达和鉴定及其抗真菌潜力的研究

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-30 DOI: 10.1016/j.pep.2024.106658

Mansi Jani, Komal K. Sapara, Darshan Dharajiya, Amrutlal K. Patel, Chaitanya Joshi

{"title":"Isolation, expression, and characterization of potato (Solanum tuberosum) GH family 17 β-1,3-glucanase (Stglu) for exploring its potential as an antifungal agent","authors":"Mansi Jani, Komal K. Sapara, Darshan Dharajiya, Amrutlal K. Patel, Chaitanya Joshi","doi":"10.1016/j.pep.2024.106658","DOIUrl":"10.1016/j.pep.2024.106658","url":null,"abstract":"<div><div>Plant glucanases, including potato glucanase, are pivotal in biological processes such as cell growth, development, and defense against pathogens. These enzymes hold substantial promises in biotechnological applications, especially genetic engineering for enhancing crop disease resistance and stress tolerance. In this study, from <em>Solanum tuberosum,</em> glycosyl hydrolases family 17 (GH-17) <em>β-1,3-glucanase</em> (<em>Stglu</em>) was cloned, expressed, characterized and its antifungal activity was evaluated. The gene was isolated from infected potato plants and cloned into the pDrive and subsequently into the pET32a (+) protein expression vector. Sequence analysis revealed a 1044 bp open reading frame encoding a 347 amino acid protein with an anticipated molecular weight of 38 kDa and a signature motif (-IEIIVSESGWPSEG-) of the GH-17 family. The recombinant β-1,3-glucanase (Stglu) protein was expressed in <em>E. coli</em> Rosetta-gami 2 (DE3) cells. After recovery from inclusion bodies using urea buffer solubilization and refolding by dialysis, expression of Stglu protein was confirmed by Western blot analysis using an anti-His antibody. Enzymatic assays were performed to characterize β-1,3-glucanase activity which showed its maximum activity at pH 7.0 and 37 °C. Plate assays for substrate specificity showed that the enzyme hydrolyzed azo-barley β-glucan and laminarin. The metal ions strongly affected the enzyme's activity; Ca<sup>2+</sup> acted as a weak activator. Plate assays further indicated the antifungal activity of Stglu against the plant pathogen <em>Fusarium solani</em>, showing a biotechnological potential tool in controlling fungal pathogenicity in crop plants.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106658"},"PeriodicalIF":1.4,"publicationDate":"2024-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142914921","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

Improving process robustness of cation exchange chromatography with cationic buffers for the reduction of aggregates 用阳离子缓冲液减少聚集体，提高阳离子交换色谱的过程稳健性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-27 DOI: 10.1016/j.pep.2024.106657

Shuo Wang, Shuaihua Wang, Dijing Shi, Ruomei Lv

引用次数: 0

Production and functional analysis of a phage displayed scFv recombinant antibody targeting EGFR/HER2 dimerization domain 靶向EGFR/HER2二聚化域的scFv重组抗体噬菌体的制备及功能分析

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-23 DOI: 10.1016/j.pep.2024.106649

Mina Dabiri , Mohsen Tehrani , Alireza Rafiei , Reza Valadan

{"title":"Production and functional analysis of a phage displayed scFv recombinant antibody targeting EGFR/HER2 dimerization domain","authors":"Mina Dabiri , Mohsen Tehrani , Alireza Rafiei , Reza Valadan","doi":"10.1016/j.pep.2024.106649","DOIUrl":"10.1016/j.pep.2024.106649","url":null,"abstract":"<div><h3>Background</h3><div>Tumor cells exploit epidermal growth factor receptor (EGFR) family to develop resistance against therapeutic antibodies, such as Herceptin. Upon ligand binding, dimerization between EGFR and HER2 is one of the most important causes of treatment failure in breast cancer and other cancers expressing EGFR and HER2. The aim of this study was to develop and evaluate the function of a human recombinant single-chain variable fragment (scFv) antibody against the dimerization domain of EGFR to inhibit its interaction with other members of the epidermal growth factor receptor family, especially HER2.</div></div><div><h3>Methods</h3><div>scFv against EGFR was expressed and purified. Cell-ELISA, MTT assay, inhibition of STAT3 phosphorylation, quantitative RT-PCR, and dimerization inhibition were performed on EGFR and HER2 expressing cell lines to characterize functional properties of the produced scFv. The conformational structure of the produced scFv and its binding ability to EGFR was computationally investigated.</div></div><div><h3>Results</h3><div><em>In vitro</em> binding analysis by cell-ELISA revealed the EGFR binding ability of the purified antibodies and confirmed by immunoblotting. ScFvs preferentially reduced the proliferation and survival of MCF7, MDA-MB-468, and SKOV3 cell lines with no effect on the VERO line. More considerably, MCF7 cells treated with the scFv antibody showed reduced STAT3 phosphorylation, decreased Bcl-2 expression, and increased Bax expression. Finally, the scFvs hindered EGFR and HER2 dimerization.</div></div><div><h3>Conclusion</h3><div>The produced scFv antibody showed to be functional in a simultaneous blockade of EGFR and HER2, suggesting its potential as a promising candidate for targeted therapy against various EGFR overexpressing tumors.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106649"},"PeriodicalIF":1.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142897108","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

Development of a novel capture step for purification of antigen binding fragments (Fabs) 抗原结合片段（fab）纯化新捕获步骤的开发。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-17 DOI: 10.1016/j.pep.2024.106647

Anupa Anupa , Anurag S. Rathore

引用次数: 0