Protein expression and purification最新文献_第2页

Simplified purification process of AsnB3 deamidated insulin variant and its comparison with human insulin

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-26 DOI: 10.1016/j.pep.2025.106665

Chinnappa Reddy V , Koduru Srivatsa , Navratna Vajpai , Ganeshan R , Aditya Upadhya , Apoorva Srivastava , Somesh B P , Kishore K.R. Tetala , Ashutosh Kumar , Partha Hazra

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Expression, characterization and anti-colon cancer activity of recombinant ginseng peptides with amino acid tandem repeats

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-23 DOI: 10.1016/j.pep.2025.106663

Yu Feng , Weina Li , Weigang Yuwen , Ru Xu , Chenhui Zhu , Zhiguang Duan , Daidi Fan

{"title":"Expression, characterization and anti-colon cancer activity of recombinant ginseng peptides with amino acid tandem repeats","authors":"Yu Feng , Weina Li , Weigang Yuwen , Ru Xu , Chenhui Zhu , Zhiguang Duan , Daidi Fan","doi":"10.1016/j.pep.2025.106663","DOIUrl":"10.1016/j.pep.2025.106663","url":null,"abstract":"<div><div>Ginseng peptides, small molecule active ingredients in ginseng, are mainly extracted naturally or synthesized chemically, but high costs and difficulties hinder further research. In this study, a ginseng hexapeptide FKEHGY, named antitumor peptide 0601 (AT0601) and its five tandem sequence repeats AT0605, were expressed in <em>Bacillus subtilis</em> WB600 for the first time, and the bioactivity study showed that the anticancer activity of AT0605 was even significantly higher than that of AT0601 for colon cancer CT26 cells, with IC50s of 16.82 ± 1.3 μM (48 h) and 855.57 ± 6.04 μM (48 h), respectively, i.e. AT0605 achieves the same inhibition rate for CT26 cells at a concentration 50 times lower than that of AT0601. Similar to the ginseng peptide AT0601, recombinant AT0605 also inhibited cell growth by blocking cells in the G1 phase and activated the mitochondria-associated caspase pathway to induce apoptosis. In conclusion, all two kinds of the recombinant ginseng peptides could also inhibit cell proliferation through mechanisms such as inhibiting cell cycle arrest and inducing a decrease in mitochondrial membrane potential.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106663"},"PeriodicalIF":1.4,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143041299","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

Bacterial expression, purification and characterization of the human Dectin-1 lectin domain for structural and functional studies

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-23 DOI: 10.1016/j.pep.2025.106668

Hanako Chiba , Noriyoshi Manabe , Junko Naito , Norihisa Nishida , Naohito Ohno , Yoshiki Yamaguchi

{"title":"Bacterial expression, purification and characterization of the human Dectin-1 lectin domain for structural and functional studies","authors":"Hanako Chiba , Noriyoshi Manabe , Junko Naito , Norihisa Nishida , Naohito Ohno , Yoshiki Yamaguchi","doi":"10.1016/j.pep.2025.106668","DOIUrl":"10.1016/j.pep.2025.106668","url":null,"abstract":"<div><div>Dectin-1 (CLEC7A), a C-type lectin-like receptor that recognizes β-1,3 glucans, has a key role in the innate immune system. While the lectin domain of mouse Dectin-1 has been solubilized and refolded from inclusion bodies in <em>Escherichia coli</em>, similar refolding of the human Dectin-1 lectin domain is hindered by the formation of misfolded multimers with aberrant intermolecular disulfide bonds. The aim of this study was to develop a method for the large-scale production of the human Dectin-1 lectin domain. Based on a protocol for the murine domain, the human Dectin-1 lectin domain was expressed as a fusion protein with Protein G B1, a solubility-enhancing tag. The refolding and purification conditions were then optimized by testing a range of buffers with and without Ca<sup>2+</sup> ions. The inclusion of 1 mM Ca<sup>2+</sup> ions in both the refolding and purification buffers resulted in high yields of a monomeric form of the human Dectin-1 lectin. The resulting recombinant protein was demonstrated to be functional, showing specific binding to the β-glucan laminarin as verified by thermal shift assays, gel filtration chromatography and NMR. Furthermore, NMR experiments revealed that the human Dectin-1 lectin domain binds Ca<sup>2+</sup> ions. The recombinant protein will support structural biology studies to clarify differences in β-glucan binding specificity between human and mouse Dectin-1, and to explore the effects of mutations on the functionality of human Dectin-1.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106668"},"PeriodicalIF":1.4,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143041295","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

Efficient production of recombinant human FVII in CHO cells using the piggyBac transposon system

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2025-01-22 DOI: 10.1016/j.pep.2025.106666

Zhen Yang , Xueyun Wang , Shan Luo , Hui Li , Jiangbo Xu , Linlin Liang , Zhimin He , Guangyuan Wang , Zhuobin Wu , Nan Zhong , Haijun Xiang , Zhan Zhang , Caiping Guo , Yunjia Zhang , Fei Yan

{"title":"Efficient production of recombinant human FVII in CHO cells using the piggyBac transposon system","authors":"Zhen Yang , Xueyun Wang , Shan Luo , Hui Li , Jiangbo Xu , Linlin Liang , Zhimin He , Guangyuan Wang , Zhuobin Wu , Nan Zhong , Haijun Xiang , Zhan Zhang , Caiping Guo , Yunjia Zhang , Fei Yan","doi":"10.1016/j.pep.2025.106666","DOIUrl":"10.1016/j.pep.2025.106666","url":null,"abstract":"<div><div>As an important coagulation factor, activated coagulation factor VII (FVIIa) is mainly used to treat the bleeding of hemophilia patients who have developed inhibitory antibodies against FVIII and FIX conventional treatment. Recombinant human factor VII (rhFVII) produced in mammalian cell lines have been developed as the most important resource of FVIIa. However, cell lines express rhFVII protein derived from an exogenous expression vector at a lower level than most other proteins. In the current study, we have shown efficient rhFVII production in CHO cell lines using piggyBac (PB) transposon system. rhFVII is successfully expressed in fed-batch culture of CHO cells, and the expression of rhFVII up to 100 mg/L. Moreover, the purified secreted rhFVII was determined by SDS-PAGE and Western Blot. The coagulation activity was determined by the chromogenic Activity ELISA kit. In conclusion, this study has demonstrated that the piggyBac transposon system can be used for an efficient production of recombinant FVII.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"229 ","pages":"Article 106666"},"PeriodicalIF":1.4,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143028957","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 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