Protein expression and purification最新文献_第3页

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

A simple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies 从包涵体中高效纯化重组人胰岛素的简单冻融法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-15 DOI: 10.1016/j.pep.2024.106645

S. Rajesh , Swaraj Jathar , Reema Banarjee , Monika Sharma , Shivani Palkar , S Shiva Shankar , Mahesh J. Kulkarni

{"title":"A simple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies","authors":"S. Rajesh , Swaraj Jathar , Reema Banarjee , Monika Sharma , Shivani Palkar , S Shiva Shankar , Mahesh J. Kulkarni","doi":"10.1016/j.pep.2024.106645","DOIUrl":"10.1016/j.pep.2024.106645","url":null,"abstract":"<div><div>Insulin is a pivotal peptide hormone essential for regulating glucose homeostasis. It has been known for over 100 years, but its production and purification methods are still under improvement. <em>Escherichia coli</em> based bacterial expression system is primarily used for insulin production. The human insulin protein expressed in bacteria usually forms inclusion bodies, complicating the purification process. Traditionally, insulin purification is a time-consuming process involving urea-based denaturation methods, and various refolding techniques, followed by extensive chromatographic methods. Here, we report an easy and efficient purification of human proinsulin involving freeze-thaw based solubilization method. The extracted proinsulin inclusion bodies are treated with different concentrations of urea, followed by a freeze-thaw based solubilization. The freezing was carried out at various temperatures, mainly −80 °C, −20 °C, and −196 °C to determine the optimum condition for solubilization. Highest solubilization of proinsulin from the inclusion body was achieved with 0.5M urea and −20 °C. Further Nickel NTA-based purification was performed, and the purified protein was characterized for disulfide mapping by high-resolution mass spectrometer (HRMS). We also performed glucose uptake assays to validate the functional properties of purified proinsulin. This freeze-thaw based mild solubilization approach is a fast and effective method for getting bioactive proinsulin, which will help further design better purification and processing strategies for insulin production.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106645"},"PeriodicalIF":1.4,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142838929","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

Improvements in large-scale production of tobacco etch virus protease 烟草蚀刻病毒蛋白酶大规模生产的改进。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-15 DOI: 10.1016/j.pep.2024.106648

Simon Messing, Kirsten Barnhart, Matthew Drew, Natalie Granato-Guerrero, Carissa Grose, Brianna Higgins, Min Hong, Jenna Hull, Shelley Perkins, Ivy Poon, Nitya Ramakrishnan, Amanda Seabolt, Troy Taylor, Vanessa E. Wall, Nicholas Wright, William Gillette, Dominic Esposito

{"title":"Improvements in large-scale production of tobacco etch virus protease","authors":"Simon Messing, Kirsten Barnhart, Matthew Drew, Natalie Granato-Guerrero, Carissa Grose, Brianna Higgins, Min Hong, Jenna Hull, Shelley Perkins, Ivy Poon, Nitya Ramakrishnan, Amanda Seabolt, Troy Taylor, Vanessa E. Wall, Nicholas Wright, William Gillette, Dominic Esposito","doi":"10.1016/j.pep.2024.106648","DOIUrl":"10.1016/j.pep.2024.106648","url":null,"abstract":"<div><div>Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms. During our work we detected post-translational modification (gluconoylation and phosphogluconoylation) of TEV protease and the subsequent effects this has on the purity of the protein. Subsequently we made our <em>pgl</em> plus bacteria that negates these modifications and their effects. We also introduce a GFP-based assay for measurement of activity and ultimately a new set of protocols for producing 400–500 mg/L TEV protease.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"228 ","pages":"Article 106648"},"PeriodicalIF":1.4,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142838932","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

Heterologous expression, purification and generation of specific antibodies for Annexin A8 异源表达、纯化和生成 Annexin A8 的特异性抗体。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-12 DOI: 10.1016/j.pep.2024.106644

Zexin Lin , Wei Sun , Xuemei Zhao , Yang Chen , Kerry M. Loomes , Hongbo Li , Hannah Xiaoyan Hui , Donghai Wu

{"title":"Heterologous expression, purification and generation of specific antibodies for Annexin A8","authors":"Zexin Lin , Wei Sun , Xuemei Zhao , Yang Chen , Kerry M. Loomes , Hongbo Li , Hannah Xiaoyan Hui , Donghai Wu","doi":"10.1016/j.pep.2024.106644","DOIUrl":"10.1016/j.pep.2024.106644","url":null,"abstract":"<div><div>Annexin A8 (ANXA8) is a member of the Annexin gene family, with high levels of its mRNA and protein observed in various tumor tissues, making it a potential tumor biomarker. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) to specifically detect the presence and accurately determine the concentration of ANXA8. To this end, we expressed a series of proteins using both <em>Pichia pastoris</em> and <em>Escherichia coli</em> expression systems. Particularly, human ANXA8 expressed in <em>Pichia pastoris</em> was used as the antigen and for antibody screening, while human ANXA2 and ANXA5 expressed in <em>Pichia pastoris</em> and murine ANXA8 expressed in <em>E. coli</em> BL21 (DE3) were used as counter screens to eliminate cross-reactive antibodies and to select antibodies that show specificity to ANXA8 from both human and mouse. Through research efforts with production and purification of recombinant proteins, immunization, specificity screening and selection of epitope pairing, two specific monoclonal antibodies, E9 and B7, apparently targeting different epitopes of ANXA8, were obtained. The specificity of the antibody pair was checked against recombinant human ANXA2 and ANXA5 with ANXA8 as the positive control. Western Blot and Sandwich ELISA demonstrate superb sensitivity and specificity with a detection limit of about 0.065 ng/mL for the latter. In summary, this study provides an experimental tool for the quantitative determination of ANXA8 concentration and a potential tumor biomarker to monitor disease progression.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106644"},"PeriodicalIF":1.4,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142823761","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

MabSelect VH3 Protein A affinity resin effectively separates antibody species containing different numbers of VH3 domain and shows improved aggregate separation capability MabSelect VH3 蛋白 A 亲和树脂能有效分离含有不同数量 VH3 结构域的抗体种类，并显示出更强的聚集分离能力。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-12 DOI: 10.1016/j.pep.2024.106646

Wanyuan Dong, Rongrong Wang, Yifeng Li

{"title":"MabSelect VH3 Protein A affinity resin effectively separates antibody species containing different numbers of VH3 domain and shows improved aggregate separation capability","authors":"Wanyuan Dong, Rongrong Wang, Yifeng Li","doi":"10.1016/j.pep.2024.106646","DOIUrl":"10.1016/j.pep.2024.106646","url":null,"abstract":"<div><div>MabSelect VH3 is a new Protein A resin recently launched by Cytiva. According to the manufacturer, the Protein A ligand of MabSelect VH3 has been engineered to disrupt and reinforce its Fc and VH3 binding capabilities, respectively. Thus, different from regular Protein A resins, this new Protein A resin has affinity for VH3 domain only. The vendor has suggested that MabSelect VH3, owing to its unique selectivity, can separate byproducts that are different from the product in the number of VH3 domain. In the current work, with two concrete cases, we demonstrated that MabSelect VH3 indeed allows effective separation of species containing different numbers of VH3 domain. In addition, we showed that, in comparison to regular Protein A resins, MabSelect VH3 also exhibits improved aggregate separation potential. Thus, for cases where product and byproduct differ in the number of VH3 domain and/or culture harvest contains high percentage of aggregates, MabSelect VH3 is a better alternative than regular Protein A for product capture as it allows simultaneous removal of byproducts and aggregates.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106646"},"PeriodicalIF":1.4,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142823762","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