Protein expression and purification最新文献_第8页

Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis GFP 靶向纳米抗体的工程化和表征：表达、纯化和翻译后修饰分析。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-21 DOI: 10.1016/j.pep.2024.106501

Dunchu Weng, Lin Yang, Yajun Xie

{"title":"Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis","authors":"Dunchu Weng, Lin Yang, Yajun Xie","doi":"10.1016/j.pep.2024.106501","DOIUrl":"10.1016/j.pep.2024.106501","url":null,"abstract":"<div><p>Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from <em>Escherichia coli</em> at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106501"},"PeriodicalIF":1.6,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141088531","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

Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production 重组人白细胞介素-2 在大肠杆菌中的可溶性表达及其简便生产。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-20 DOI: 10.1016/j.pep.2024.106507

Minhui Zhang , Yongxiang Zheng , Sa Wang, Pengyu Wang, Jingbei Huang, Xiaotong Song, Rong Yu, Chun Zhang

{"title":"Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production","authors":"Minhui Zhang , Yongxiang Zheng , Sa Wang, Pengyu Wang, Jingbei Huang, Xiaotong Song, Rong Yu, Chun Zhang","doi":"10.1016/j.pep.2024.106507","DOIUrl":"10.1016/j.pep.2024.106507","url":null,"abstract":"<div><p>Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in <em>E. coli</em> due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in <em>E. coli</em> Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106507"},"PeriodicalIF":1.6,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141082119","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 NP-hoc fusion protein: Utilizing influenza a nucleoprotein and phage T4 hoc protein 一种 NP-Hoc 融合蛋白的表达和纯化：利用甲型流感核蛋白和噬菌体 T4 Hoc 蛋白。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-19 DOI: 10.1016/j.pep.2024.106506

Francisco de Jesús Balderas-Cisneros , Angel León-Buitimea , Xristo Zarate , José Rubén Morones-Ramírez

{"title":"Expression and purification of an NP-hoc fusion protein: Utilizing influenza a nucleoprotein and phage T4 hoc protein","authors":"Francisco de Jesús Balderas-Cisneros , Angel León-Buitimea , Xristo Zarate , José Rubén Morones-Ramírez","doi":"10.1016/j.pep.2024.106506","DOIUrl":"10.1016/j.pep.2024.106506","url":null,"abstract":"<div><p>Influenza poses a substantial health risk, with infants and the elderly being particularly susceptible to its grave impacts. The primary challenge lies in its rapid genetic evolution, leading to the emergence of new Influenza A strains annually. These changes involve punctual mutations predominantly affecting the two main glycoproteins: Hemagglutinin (HA) and Neuraminidase (NA). Our existing vaccines target these proteins, providing short-term protection, but fall short when unexpected pandemics strike. Delving deeper into Influenza's genetic makeup, we spotlight the nucleoprotein (NP) - a key player in the transcription, replication, and packaging of RNA. An intriguing characteristic of the NP is that it is highly conserved across all Influenza A variants, potentially paving the way for a more versatile and broadly protective vaccine. We designed and synthesized a novel NP-Hoc fusion protein combining Influenza A nucleoprotein and T4 phage Hoc, cloned using Gibson assembly in <em>E. coli</em>, and purified via ion affinity chromatography. Simultaneously, we explore the T4 coat protein Hoc, typically regarded as inconsequential in controlled viral replication. Yet, it possesses a unique ability: it can link with another protein, showcasing it on the T4 phage coat. Fusing these concepts, our study designs, expresses, and purifies a novel fusion protein named NP-Hoc. We propose this protein as the basis for a new generation of vaccines, engineered to guard broadly against Influenza A. The excitement lies not just in the immediate application, but the promise this holds for future pandemic resilience, with NP-Hoc marking a significant leap in adaptive, broad-spectrum influenza prevention.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106506"},"PeriodicalIF":1.6,"publicationDate":"2024-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141076404","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

Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors 在 WAVE 生物反应器中利用外显子稳定池快速、稳健地生产重组蛋白。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-18 DOI: 10.1016/j.pep.2024.106505

Melanie Dannemeyer, Anna Berling, Sara Kanje, Henric Enstedt, LanLan Xu, Delaram Afshari, Malin Westin, Gabriella Hober, Mathias Uhlén, Sophia Hober, Hanna Tegel

{"title":"Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors","authors":"Melanie Dannemeyer, Anna Berling, Sara Kanje, Henric Enstedt, LanLan Xu, Delaram Afshari, Malin Westin, Gabriella Hober, Mathias Uhlén, Sophia Hober, Hanna Tegel","doi":"10.1016/j.pep.2024.106505","DOIUrl":"10.1016/j.pep.2024.106505","url":null,"abstract":"<div><p>Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106505"},"PeriodicalIF":1.6,"publicationDate":"2024-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824000779/pdfft?md5=9c8b668160a3752d0db74721d52fe3ff&pid=1-s2.0-S1046592824000779-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141071895","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

CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability. CaptureSelect FcXP 亲和培养基具有很强的聚合分离能力。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-15 DOI: 10.1016/j.pep.2024.106503

Wanyuan Dong, Dan Zhang, Yifeng Li

引用次数: 0

Production of VP3-only virus-like particles of Adeno-associated virus 2 in E. coli cells 在大肠杆菌细胞中生产仅有 VP3 的腺相关病毒 2 病毒样颗粒。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-15 DOI: 10.1016/j.pep.2024.106502

Chengyu Fu, Shruthi Gobbooru , Ashley T. Martino, Woon-Kai Low

引用次数: 0

CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability CaptureSelect FcXP 亲和培养基具有很强的聚合分离能力。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-15 DOI: 10.1016/j.pep.2024.106503

Wanyuan Dong, Dan Zhang, Yifeng Li

引用次数: 0

A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications 一种用于 CRISPR 干扰/激活 (CRISPRi/a) 的 dCas9 强健表达和一步纯化的新方法。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-07 DOI: 10.1016/j.pep.2024.106500

Harshita Pandey , Binduma Yadav , Koushik Shah , Raminder Kaur , Diksha Choudhary , Nishtha Sharma , Vikas Rishi

{"title":"A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications","authors":"Harshita Pandey , Binduma Yadav , Koushik Shah , Raminder Kaur , Diksha Choudhary , Nishtha Sharma , Vikas Rishi","doi":"10.1016/j.pep.2024.106500","DOIUrl":"10.1016/j.pep.2024.106500","url":null,"abstract":"<div><p>CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. <em>In vitro</em> Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an <em>E. coli</em>-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different <em>in vitro</em> and <em>in vivo</em> studies.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"220 ","pages":"Article 106500"},"PeriodicalIF":1.6,"publicationDate":"2024-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140892302","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

An important consideration when expressing mAbs in Escherichia coli 在大肠杆菌中表达 mAbs 是一个重要的考虑因素。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-03 DOI: 10.1016/j.pep.2024.106499

William B. Siegall , Rachel B. Lyon , Zvi Kelman

引用次数: 0

Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater 从纸浆和造纸厂废水中分离的枯草芽孢杆菌菌株中提取的新型 β-1,4-内切葡聚糖酶的表达和表征

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-01 DOI: 10.1016/j.pep.2024.106490

Joel Ríos-Alvarado , Olga Noelia Avitia-Rodríguez , Norma Urtiz-Estrada , David Enrique Zazueta-Álvarez , Javier López-Miranda , Perla Guadalupe Vázquez-Ortega , Juan Antonio Rojas-Contreras

{"title":"Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater","authors":"Joel Ríos-Alvarado , Olga Noelia Avitia-Rodríguez , Norma Urtiz-Estrada , David Enrique Zazueta-Álvarez , Javier López-Miranda , Perla Guadalupe Vázquez-Ortega , Juan Antonio Rojas-Contreras","doi":"10.1016/j.pep.2024.106490","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106490","url":null,"abstract":"<div><p>The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a <em>Bacillus subtilis</em> strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (<em>eglS</em>) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed <em>E. coli</em> BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"220 ","pages":"Article 106490"},"PeriodicalIF":1.6,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140825305","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