Protein expression and purification最新文献_第7页

Insights into the association of the Chlamydia trachomatis type III secretion chaperone complex, Scc4:Scc1, from sequential expression in Escherichia coli 通过在大肠杆菌中的连续表达揭示沙眼衣原体 III 型分泌伴侣复合体 Scc4:Scc1 的关联。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-08 DOI: 10.1016/j.pep.2024.106532

Hemanthie C. Wickramasinghe , Juliette N. Lincoln , Anne E. D'Armond , Sadie A. Noble , Li Shen , Megan A. Macnaughtan

{"title":"Insights into the association of the Chlamydia trachomatis type III secretion chaperone complex, Scc4:Scc1, from sequential expression in Escherichia coli","authors":"Hemanthie C. Wickramasinghe , Juliette N. Lincoln , Anne E. D'Armond , Sadie A. Noble , Li Shen , Megan A. Macnaughtan","doi":"10.1016/j.pep.2024.106532","DOIUrl":"10.1016/j.pep.2024.106532","url":null,"abstract":"<div><p><em>Chlamydia trachomatis</em> (<em>CT</em>) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and <em>Chlamydia</em>. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in <em>CT</em>'s developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 <em>in vitro</em> to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the <em>in vitro</em> association studies suggest that partial protein denaturation and/or components in <em>E. coli</em> lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in <em>E. coli</em> under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation <em>in vivo</em>. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous <em>in vitro</em> association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in <em>E. coli</em>.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106532"},"PeriodicalIF":1.6,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141301445","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 novel method for synthesizing authentic SARS-CoV-2 main protease 合成正宗 SARS-CoV-2 主要蛋白酶的新方法。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-08 DOI: 10.1016/j.pep.2024.106531

Cheng Zhao, Yi Rong, Shuyuan Shi, Wen-chao Gao, Chaofeng Zhang

{"title":"A novel method for synthesizing authentic SARS-CoV-2 main protease","authors":"Cheng Zhao, Yi Rong, Shuyuan Shi, Wen-chao Gao, Chaofeng Zhang","doi":"10.1016/j.pep.2024.106531","DOIUrl":"10.1016/j.pep.2024.106531","url":null,"abstract":"<div><p>The SARS-CoV-2 main protease (M<sup>pro</sup>) plays a crucial role in virus amplification and is an ideal target for antiviral drugs. Currently, authentic M<sup>pro</sup> is prepared through two rounds of proteolytic cleavage. In this method, M<sup>pro</sup> carries a self-cleavage site at the N-terminus and a protease cleavage site followed by an affinity tag at the C-terminus. This article proposes a novel method for producing authentic M<sup>pro</sup> through single digestion. M<sup>pro</sup> was constructed by fusing a His tag containing TEV protease cleavage sites at the N-terminus. The expressed recombinant protein was digested by TEV protease, and the generated protein had a decreased molecular weight and significantly increased activity, which was consistent with that of authentic M<sup>pro</sup> generated by the previous method. These findings indicated that authentic M<sup>pro</sup> was successfully obtained. Moreover, the substrate specificity of M<sup>pro</sup> was investigated. M<sup>pro</sup> had a strong preference for Phe at position the P2, which suggested that the S2 subsite was an outstanding target for designing inhibitors. This article also provides a reference for the preparation of M<sup>pro</sup> for sudden coronavirus infection in the future.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106531"},"PeriodicalIF":1.6,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296628","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

Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicago sativa 释放与志贺氏杆菌痢疾杆菌 IpaDSTxB 融合的延展素信号肽和弹性蛋白样多肽标签的潜力，以改善烟草和麦角草中蛋白质的表达和纯化。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-07 DOI: 10.1016/j.pep.2024.106521

AmirMohammad Soleimani, Houshang Alizadeh

{"title":"Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicago sativa","authors":"AmirMohammad Soleimani, Houshang Alizadeh","doi":"10.1016/j.pep.2024.106521","DOIUrl":"10.1016/j.pep.2024.106521","url":null,"abstract":"<div><p>Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed <em>Shigella dysenteriae</em>'s IpaDSTxB fused to both NtEXT and ELP in both <em>Nicotiana tabacum</em> and <em>Medicago sativa</em>. Our results demonstrated that <em>N. tabacum</em>, with an average yield of 6.39 ng/μg TSP, outperforms <em>M. sativa</em>, which had an average yield of 3.58 ng/μg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in <em>N. tabacum</em> and <em>M. sativa</em>, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both <em>N. tabacum</em> and <em>M. sativa</em> by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106521"},"PeriodicalIF":1.6,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296629","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

Screening, expression and anti-tumor functional identification of anti-LAG-3 nanobodies 抗 LAG-3 纳米抗体的筛选、表达和抗肿瘤功能鉴定。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-06 DOI: 10.1016/j.pep.2024.106522

Dan Jiang , Rui Chen , Liyan Wang , Guangxian Xu

{"title":"Screening, expression and anti-tumor functional identification of anti-LAG-3 nanobodies","authors":"Dan Jiang , Rui Chen , Liyan Wang , Guangxian Xu","doi":"10.1016/j.pep.2024.106522","DOIUrl":"10.1016/j.pep.2024.106522","url":null,"abstract":"<div><h3>Objective</h3><p>To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells.</p></div><div><h3>Methods</h3><p>Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjμgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc.</p></div><div><h3>Results</h3><p>One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 μg/mL.</p></div><div><h3>Conclusion</h3><p>The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106522"},"PeriodicalIF":1.6,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141293649","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 characterization of CTP synthase PyrG in Staphylococcus aureus 金黄色葡萄球菌中 CTP 合成酶 PyrG 的表达、纯化和表征

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-03 DOI: 10.1016/j.pep.2024.106520

Dafeng Liu , Zhu Tian , Kuerban Tusong , Hayrinsa Mamat , Yihan Luo

{"title":"Expression, purification and characterization of CTP synthase PyrG in Staphylococcus aureus","authors":"Dafeng Liu , Zhu Tian , Kuerban Tusong , Hayrinsa Mamat , Yihan Luo","doi":"10.1016/j.pep.2024.106520","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106520","url":null,"abstract":"<div><p><em>Staphylococcus aureus</em> (<em>S. aureus</em>) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates <em>S. aureus</em> infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against <em>S. aureus</em> due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of <em>S. aureus</em> PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of <em>S. aureus</em> to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against <em>S. aureus</em> infections.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106520"},"PeriodicalIF":1.6,"publicationDate":"2024-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141242756","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

Metal binding feature of copper‒induced metallothionein from freshwater crab Sinopotamon henanense reveals its Cu‒thionein character 河蟹铜诱导金属硫蛋白的金属结合特征揭示了其铜硫蛋白特性

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-01 DOI: 10.1016/j.pep.2024.106519

Huizhen Yang , Ziyan Zhao , Hongquan Li , Lan Wang

{"title":"Metal binding feature of copper‒induced metallothionein from freshwater crab Sinopotamon henanense reveals its Cu‒thionein character","authors":"Huizhen Yang , Ziyan Zhao , Hongquan Li , Lan Wang","doi":"10.1016/j.pep.2024.106519","DOIUrl":"10.1016/j.pep.2024.106519","url":null,"abstract":"<div><p><em>Sinopotamon Henanense</em> expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in <em>Escherichia coli</em> and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in <em>S. Henanense</em>.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106519"},"PeriodicalIF":1.6,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141236993","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

Corrigendum to “Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater” [J. Protein Expr. Purif., 220 (2024) 106490] 更正："从纸浆和造纸厂废水中分离出的枯草芽孢杆菌菌株的新型β-1,4-内切葡聚糖酶的表达和表征" [J. Protein Expr. Purif.，220 (2024) 106490]。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-30 DOI: 10.1016/j.pep.2024.106517

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

引用次数: 0

Biosynthesis of α-keto acids and resolution of chiral amino acids by l-amino acid deaminases from Proteus mirabilis 奇异变形杆菌 L-氨基酸脱氨酶的 α-酮酸生物合成和手性氨基酸的解析。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-29 DOI: 10.1016/j.pep.2024.106518

Junzhang Chang , Yuxin Zhang , Zhiwei Li , Yunfeng Ma , Xueqin Hu , Jingwen Yang , Hongbin Zhang

{"title":"Biosynthesis of α-keto acids and resolution of chiral amino acids by l-amino acid deaminases from Proteus mirabilis","authors":"Junzhang Chang , Yuxin Zhang , Zhiwei Li , Yunfeng Ma , Xueqin Hu , Jingwen Yang , Hongbin Zhang","doi":"10.1016/j.pep.2024.106518","DOIUrl":"10.1016/j.pep.2024.106518","url":null,"abstract":"<div><p>Chiral amino acids and their deamination products, α-keto acids, have important applications in food, medicine, and fine chemicals. In this study, two <span>l</span>-amino acid deaminase genes from <em>Proteus mirabilis</em>, PM473 of type Ⅰ and PM471 of type Ⅱ were cloned and expressed in <em>Escherichia coli</em> respectively, expected to achieve the chiral separation of amino acids. Extensive substrate preference testing showed that both deaminases had catalytic effects on the <span>d</span>-amino acid component of the D, <span>l</span>-amino acids, and PM473 has a wider catalytic range for amino acids. When D, L-Cys was used as the substrate, all L-Cys components and 75.1 % of D-Cys were converted to mercapto pyruvate, and the remaining D-Cys was a single chiral enantiomer. Molecular docking analysis showed that the interaction between the substrate and the key residues affected the stereoselectivity of enzymes. The compatibility of hydrophobicity between the binding pocket and substrate may be the basic factor that affects the substrate selectivity. This work provides an alternative method for the production of α-keto acids and the resolution of chiral amino acids.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106518"},"PeriodicalIF":1.6,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141184398","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

CUREs for high-level Galectin-3 expression CUREs for High-Level Galectin-3 Expression.

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-25 DOI: 10.1016/j.pep.2024.106516

Alexander A. Charbonneau, Elizabeth J. Reicks, John F. Cambria, Jacob Inman, Daria Danley, Emmie A. Shockley, Ravenor Davion, Isabella Salgado, Erienne G. Norton , Lucy J. Corbett, Lucy E. Hanacek, Jordan G. Jensen, Marguerite A. Kibodeaux, Tess K. Kirkpatrick, Keilen M. Rausch, Samantha R. Roth, Bernadette West, Kenai E. Wilson, C. Martin Lawrence, Mary J. Cloninger

{"title":"CUREs for high-level Galectin-3 expression","authors":"Alexander A. Charbonneau, Elizabeth J. Reicks, John F. Cambria, Jacob Inman, Daria Danley, Emmie A. Shockley, Ravenor Davion, Isabella Salgado, Erienne G. Norton , Lucy J. Corbett, Lucy E. Hanacek, Jordan G. Jensen, Marguerite A. Kibodeaux, Tess K. Kirkpatrick, Keilen M. Rausch, Samantha R. Roth, Bernadette West, Kenai E. Wilson, C. Martin Lawrence, Mary J. Cloninger","doi":"10.1016/j.pep.2024.106516","DOIUrl":"10.1016/j.pep.2024.106516","url":null,"abstract":"<div><p>Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds β-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, <em>E. coli</em> is employed as a heterologous expression system, and galectin expression is induced with isopropyl β-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106516"},"PeriodicalIF":1.6,"publicationDate":"2024-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141156997","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 single step efficient refolding of recombinant tissue plasminogen activator (Reteplase) from E. coli 大肠杆菌重组组织纤溶酶原激活剂（Reteplase）的异源表达、纯化和单步高效重折叠。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-05-22 DOI: 10.1016/j.pep.2024.106504

Meha Bhatt, Haidar Abbas Masi, Amrutlal Patel, Niraj Kumar Singh, Chaitanya Joshi

{"title":"Heterologous expression, purification and single step efficient refolding of recombinant tissue plasminogen activator (Reteplase) from E. coli","authors":"Meha Bhatt, Haidar Abbas Masi, Amrutlal Patel, Niraj Kumar Singh, Chaitanya Joshi","doi":"10.1016/j.pep.2024.106504","DOIUrl":"10.1016/j.pep.2024.106504","url":null,"abstract":"<div><p>Reteplase (recombinant plasminogen activator, rPA) is a mutant non-glycosylated tissue-type plasminogen activator (tPA) containing 355 amino acids with longer half-life and promising thrombolytic activity than its original counterpart, full length tPA. In this study, we aimed to produce and optimize the purification process of recombinant tissue-type plasminogen activator (tPA) known as Reteplase (rPA). Reteplase cDNA synthesized from total mRNA isolated from human placenta was PCR amplified, cloned into a pET-28a(+) <em>E. coli</em> expression vector and expressed in Rosetta-gami 2 <em>E. coli</em> (Novagen<sup>Ⓡ</sup>) host. rPA was expressed as an inclusion body in <em>E. coli</em> and its biological activity was achieved after single step solubilization, purification and refolding. We exploited the strategy of Slow Refolding using Gradual Dialysis (SRGD) in which a refolding buffer containing glutathione oxidized (1 mM GSSG) and glutathione reduced (3 mM GSH) and pH 9.0 was used. Using the SRGD method, we were able to successfully obtain the protein in its active form. We obtained 4.26 mg of active refolded protein from a 50 mL culture that was scaled up in a bioreactor. The purity and homogeneity of rPA was evaluated by SDS-PAGE, Western blotting and mass spectrometry. Circular dichroism spectroscopy was conducted to evaluate the refolding and stability of the refolded rPA in comparison to reference standard rPA. The thrombolytic potential of rPA was assessed by fibrin plate assay and <em>In Vitro</em> clot lysis assay. The presented protocol offers a viable approach for enhancing both the yield and refolding efficiency of reteplase, potentially resulting in an increase in yield.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"221 ","pages":"Article 106504"},"PeriodicalIF":1.6,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141088577","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