Protein expression and purification最新文献_第5页

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

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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}

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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}

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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

Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells 人苦味受体TAS2R14在HEK293细胞中功能表达的优化载体。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-10 DOI: 10.1016/j.pep.2024.106643

Christine Belloir, Adèle Gautier, Adeline Karolkowski, Thomas Delompré, Mathilde Jeannin, Lucie Moitrier, Fabrice Neiers, Loïc Briand

{"title":"Optimized vector for functional expression of the human bitter taste receptor TAS2R14 in HEK293 cells","authors":"Christine Belloir, Adèle Gautier, Adeline Karolkowski, Thomas Delompré, Mathilde Jeannin, Lucie Moitrier, Fabrice Neiers, Loïc Briand","doi":"10.1016/j.pep.2024.106643","DOIUrl":"10.1016/j.pep.2024.106643","url":null,"abstract":"<div><div>Bitter is one of the five basic taste qualities, along with salty, sour, sweet and umami, used by mammals to access the quality of their food and orient their eating behaviour. Bitter taste detection prevents the ingestion of food potentially contaminated by bitter-tasting toxins. Bitter taste perception is mediated by a family of G protein-coupled receptors (GPCRs) called TAS2Rs. Humans possess 25 TAS2Rs (human type II taste receptors), enabling the detection of thousands of chemically diverse bitter compounds. The identification of agonists/antagonists and molecular mechanisms that govern receptor-ligand interaction has been primarily achieved through functional expression of TAS2Rs in heterologous cells. However, TAS2R receptors, like many other GPCRs, suffer from marginal cell surface expression. In this study, we compared the functionality of 9 engineered chimeric receptors, focusing our experiments on TAS2R14, a broadly tuned receptor that recognizes over 151 identified compounds. Among the different tested signal peptides, rat somatostatin receptor subtype 3 results in higher potency of aristolochic acid-induced calcium signalling than other tested export tags, such as bovine rhodopsin, murine Igκ-chain or human mGluR5. The addition of a MAX sequence enhances both TAS2R14 potency and efficacy. We also confirm that the FLAG epitope, when located at the C-terminal, interferes less with the TAS2R14 functionality, enabling reliable evaluation of this receptor at the cell surface using immunohistochemistry. Finally, these observations are also confirmed for TAS2R14 and TAS1R2/TAS1R3 (the sweet taste receptor) stimulated by 12 bitter compounds and by sucralose and neotame, respectively.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106643"},"PeriodicalIF":1.4,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142819017","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimized extraction, identification and characterization of the mosquitocidal surface layer protein from a local bacterial isolate Lysinibacillus sphaericus Q001 球形赖氨酸芽孢杆菌Q001杀蚊表层蛋白的优化提取、鉴定及特性研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-09 DOI: 10.1016/j.pep.2024.106639

Hamayun Arshad , Qurratulann Afza Gardner , Saira Ahmad , Syeda Sadia Bukhari , Muhammad Akhtar

{"title":"Optimized extraction, identification and characterization of the mosquitocidal surface layer protein from a local bacterial isolate Lysinibacillus sphaericus Q001","authors":"Hamayun Arshad , Qurratulann Afza Gardner , Saira Ahmad , Syeda Sadia Bukhari , Muhammad Akhtar","doi":"10.1016/j.pep.2024.106639","DOIUrl":"10.1016/j.pep.2024.106639","url":null,"abstract":"<div><div>Surface layer (S-layer) is an extracellular proteinous layer consisting of two-dimensional lattice. It is typically present on archaea and also found on some bacteria. S-layer proteins from some bacteria are reported to be toxic to mosquito larvae. Here, we aimed to extract and characterize the surface layer protein from a local bacterial strain named <em>Lysinibacillus sphaericus</em> Q001. This bacterium was isolated from Pakistan and characterized through various biochemical tests. It was identified as <em>Lysinibacillus sphaericus</em> through 16S rRNA ribotyping (NCBI accession no. OQ701385.1) and matrix-assisted laser desorption/ionization (MALDI) biotyping with 2.18 ± 0.059 score. The S-layer protein was extracted by both cation exchange method and guanidinium chloride extraction method. The optimized method for the extraction and purification of S-layer yielded 35 mg of protein from 1 L culture of <em>L. sphaericus</em> Q001. A potential S-layer protein band (120 kDa) detected by SDS-PAGE was confirmed by bottom-up proteomics i.e., in-gel tryptic digestion of the protein followed by MALDI-TOF analysis and peptide mass fingerprinting (PMF). The insecticidal bioassays revealed that S-layer protein of <em>L. sphaericus</em> Q001 was toxic against <em>Aedes aegypti</em> larvae with LC<sub>50</sub> value of 11 μg/ml. This shows its potential to be used as an alternative to chemical larvicides.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106639"},"PeriodicalIF":1.4,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142786779","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

Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering 纳米盘中全长流感病毒血凝素的生产和成分分析：多角度光散射的启示。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-12-07 DOI: 10.1016/j.pep.2024.106641

Tim G.J. Knetsch, Marcellus Ubbink

{"title":"Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering","authors":"Tim G.J. Knetsch, Marcellus Ubbink","doi":"10.1016/j.pep.2024.106641","DOIUrl":"10.1016/j.pep.2024.106641","url":null,"abstract":"<div><div>The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterisation by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-<em>sn</em>-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106641"},"PeriodicalIF":1.4,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142802104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0