Protein expression and purification最新文献_第9页

Removal of signal peptide variants by cation exchange chromatography: A case study 阳离子交换色谱法去除信号肽变体：案例研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-19 DOI: 10.1016/j.pep.2024.106581

Yong Fu , Yangguang Xu , Maodan Zhang, Fengjuan Lv

引用次数: 0

A multidomain PARP14 construct suitable for bacterial expression 适合细菌表达的多域 PARP14 构建物。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-17 DOI: 10.1016/j.pep.2024.106580

Constantinos Chatzicharalampous, Herwig Schüler

{"title":"A multidomain PARP14 construct suitable for bacterial expression","authors":"Constantinos Chatzicharalampous, Herwig Schüler","doi":"10.1016/j.pep.2024.106580","DOIUrl":"10.1016/j.pep.2024.106580","url":null,"abstract":"<div>Poly-ADP-ribose polymerase-14 (PARP14) can modify proteins and nucleic acids by the reversible addition of a single ADP-ribose molecule. Aberrant PARP14 functions have been related to cancer and inflammation, and its domains are involved in processes related to viral infection. Previous research indicates that PARP14 functions might be mediated via a multitude of target proteins. In vitro studies of this large multidomain enzyme have been complicated by difficulties to obtain biochemical quantities of pure protein. Here we present a strategy that allows bacterial expression and purification of a functional multidomain construct of PARP14. We substituted an internal KH domain and its neighboring unstructured region with a SUMO domain to obtain a protein construct that encompasses three macrodomains, a WWE domain, and a PARP catalytic domain. We show that the resulting construct retains both ADP-ribosyltransferase and de-MARylase activities. This construct will be useful in structural and functional studies of PARP14.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106580"},"PeriodicalIF":1.4,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001529/pdfft?md5=f6c9e5d14a9f3a631e3292300471b274&pid=1-s2.0-S1046592824001529-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142000597","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

Improved self-cleaving precipitation tags for efficient column free bioseparations 用于高效无柱生物分离的改进型自裂解沉淀标记。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-15 DOI: 10.1016/j.pep.2024.106578

Hongyu Yuan , Sai Vivek Prabhala , Michael J. Coolbaugh, Samuel D. Stimple, David W. Wood

{"title":"Improved self-cleaving precipitation tags for efficient column free bioseparations","authors":"Hongyu Yuan , Sai Vivek Prabhala , Michael J. Coolbaugh, Samuel D. Stimple, David W. Wood","doi":"10.1016/j.pep.2024.106578","DOIUrl":"10.1016/j.pep.2024.106578","url":null,"abstract":"<div>Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106578"},"PeriodicalIF":1.4,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996282","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

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells 副溶血性弧菌中的 MAM7：表达、纯化及对 RAW264.7 细胞的影响。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-15 DOI: 10.1016/j.pep.2024.106579

Qingsong Zeng, Peifang Lai, Mingqin Huang, Ximing Peng, Junjie Huang, Qintao Chen, Yanxu Chen, Huaqian Wang

{"title":"MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells","authors":"Qingsong Zeng, Peifang Lai, Mingqin Huang, Ximing Peng, Junjie Huang, Qintao Chen, Yanxu Chen, Huaqian Wang","doi":"10.1016/j.pep.2024.106579","DOIUrl":"10.1016/j.pep.2024.106579","url":null,"abstract":"<div>V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75 mM and 15 °C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7 cells, with its optimal concentration determined to be 7.5 μg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p < 0.05) in NO levels. Additionally, the relative mRNA levels of IL-1β, IL-6, and TNF-α in RAW264.7 cells were measured by qRT-PCR, showing a remarkable elevation (p < 0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7 cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106579"},"PeriodicalIF":1.4,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996283","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

Novel heterologously expressed protein, AjPSPLP-3, derived from Apostichopus japonicus exhibits cell proliferation and migration activities 异源表达的新蛋白 AjPSPLP-3 来自日本狎鸥鱼，具有细胞增殖和迁移活性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-15 DOI: 10.1016/j.pep.2024.106577

Weitao Wang , Yiwei Meng , Xin Yin , Peipei Zhao , Mengmeng Wang , Jingli Ren , Jiyuan Zhang , Lixin Zhang , Yunqian Cui , Xuekui Xia

{"title":"Novel heterologously expressed protein, AjPSPLP-3, derived from Apostichopus japonicus exhibits cell proliferation and migration activities","authors":"Weitao Wang , Yiwei Meng , Xin Yin , Peipei Zhao , Mengmeng Wang , Jingli Ren , Jiyuan Zhang , Lixin Zhang , Yunqian Cui , Xuekui Xia","doi":"10.1016/j.pep.2024.106577","DOIUrl":"10.1016/j.pep.2024.106577","url":null,"abstract":"<div>Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a Apostichopus japonicus derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine β-sheets, Cys30-Cys67, Cys38-Cys58, Cys53-Cys90, Cys56-Cys66, and Cys81-Cys102 participating in the formation of five pairs of disulfide bonds. In vitro experiments conducted on HaCaT cells proved that AjPSPLP-3 and MBP-fused AjPSPLP-3 significantly contribute to HaCaT cells proliferation and migration without exhibiting hemolytic activity on murine erythrocytes. Specifically, treatment with 10 μmol/L MBP-fused AjPSPLP-3 protein increased the viability of HaCaT cells by 12.28 % (p < 0.001), while treatment with 10 μmol/L AjPSPLP-3 protein increased viability of HaCaT cells by 6.01 % (p < 0.01). Furthermore, wound closure of MBP-fused AjPSPLP-3 and AjPSPLP-3 were 22.51 % (p < 0.01) and 7.32 % (p < 0.05) higher than that of the control groups in HaCaT cells following 24 h of incubation.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106577"},"PeriodicalIF":1.4,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996284","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

Protein inclusion into ice can dissociate subunits 蛋白质加入冰中会使亚单位解离。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-11 DOI: 10.1016/j.pep.2024.106576

Robert Eves, Peter L. Davies

引用次数: 0

Single-step purification and characterization of Pseudomonas aeruginosa azurin 铜绿假单胞菌 Azurin 的单步纯化和表征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-10 DOI: 10.1016/j.pep.2024.106566

Petra Riegerová , Matej Horváth , Filip Šebesta , Jan Sýkora , Miroslav Šulc , Antonín Vlček

{"title":"Single-step purification and characterization of Pseudomonas aeruginosa azurin","authors":"Petra Riegerová , Matej Horváth , Filip Šebesta , Jan Sýkora , Miroslav Šulc , Antonín Vlček","doi":"10.1016/j.pep.2024.106566","DOIUrl":"10.1016/j.pep.2024.106566","url":null,"abstract":"<div>Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106566"},"PeriodicalIF":1.4,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001384/pdfft?md5=8e6dd66b1b5f1cc9ef72519abe69efe9&pid=1-s2.0-S1046592824001384-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141917362","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

Biochemical characterization and antifungal activity of a recombinant β-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 来自黄杆菌 NAU1659 的重组β-1,3-葡聚糖酶 FlGluA 的生化特征和抗真菌活性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-08 DOI: 10.1016/j.pep.2024.106563

Yanxin Wang , Tingting Xie , Chenlong Ma , Yujie Zhao , Jingchen Li , Zhendong Li , Xianfeng Ye

{"title":"Biochemical characterization and antifungal activity of a recombinant β-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659","authors":"Yanxin Wang , Tingting Xie , Chenlong Ma , Yujie Zhao , Jingchen Li , Zhendong Li , Xianfeng Ye","doi":"10.1016/j.pep.2024.106563","DOIUrl":"10.1016/j.pep.2024.106563","url":null,"abstract":"<div>β-1,3-glucanases can degrade β-1,3-glucoside bonds in β-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a β-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the β-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0–8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-β-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106563"},"PeriodicalIF":1.4,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141913749","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 new alkaline pectin lyase with novel thermal and pH stability from Bacilus velezensis 来自 Velezensis 杆菌的一种具有新型热稳定性和 pH 稳定性的新型碱性果胶裂解酶。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-05 DOI: 10.1016/j.pep.2024.106564

Ze Li, Su-Yan Tian

引用次数: 0

Soluble expression of hMYDGF was improved by strain engineering and optimizations of fermentation strategies in Escherichia coli 通过菌种工程和优化大肠杆菌发酵策略，提高了 hMYDGF 的可溶性表达。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-05 DOI: 10.1016/j.pep.2024.106565

Si-yuan Su , Yong-Shan Zheng , Hui Mao , Li-bing Zhao , Man-yi Zhu , Yu-feng Yang , Ling-ting Li , Zi-ru Wang , Cheng He

{"title":"Soluble expression of hMYDGF was improved by strain engineering and optimizations of fermentation strategies in Escherichia coli","authors":"Si-yuan Su , Yong-Shan Zheng , Hui Mao , Li-bing Zhao , Man-yi Zhu , Yu-feng Yang , Ling-ting Li , Zi-ru Wang , Cheng He","doi":"10.1016/j.pep.2024.106565","DOIUrl":"10.1016/j.pep.2024.106565","url":null,"abstract":"<div>Myeloid-derived growth factor (MYDGF) is a cytokine that exhibits a variety of biological functions. This study focused on utilizing BL21(DE3) strain engineering and fermentation strategies to achieve high-level expression of soluble human MYDGF (hMYDGF) in Escherichia coli. Initially, the E. coli expressing strain BL21(DE3) was engineered by deleting the IpxM gene and inserting the GROEL/S and Trigger factor genes. The engineered E. coli strain BL21(TG)/pT-MYDGF accumulated 3557.3 ± 185.6 μg/g and 45.7 ± 6.7 mg/L of soluble hMYDGF in shake flask fermentation, representing a 15.6-fold increase compared to the control strain BL21(DE3)/pT-MYDGF. Furthermore, the yield of hMYDGF was significantly enhanced by optimizing the fermentation conditions. Under optimized conditions, the 5L bioreactor yielded up to 2665.8 ± 164.3 μg/g and 407.6 ± 42.9 mg/L of soluble hMYDGF. The results indicate that the implementation of these optimization strategies could enhance the ratio and yield of soluble proteins expressed by E.coli, thereby meeting the demands of industrial production. This study employed sophisticated strategies to lay a solid foundation for the industrial application of hMYDGF.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106565"},"PeriodicalIF":1.4,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141902719","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