Protein expression and purification最新文献_第3页

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

{"title":"Novel heterologously expressed protein, AjPSPLP-3, derived from Apostichopus japonicus exhibits cell proliferation and migration activities","authors":"","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":null,"pages":null},"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

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

{"title":"Single-step purification and characterization of Pseudomonas aeruginosa azurin","authors":"","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":null,"pages":null},"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

{"title":"Biochemical characterization and antifungal activity of a recombinant β-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659","authors":"","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":null,"pages":null},"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

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

{"title":"Soluble expression of hMYDGF was improved by strain engineering and optimizations of fermentation strategies in Escherichia coli","authors":"","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":null,"pages":null},"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

A fast and simple automated multi-step protein purification method for ÄKTA go systems 用于 ÄKTA go 系统的快速、简单的自动化多步蛋白质纯化方法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106560

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Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design 通过计算设计提高黑曲霉木聚糖酶的催化活性和热稳定性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106561

{"title":"Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design","authors":"","doi":"10.1016/j.pep.2024.106561","DOIUrl":"10.1016/j.pep.2024.106561","url":null,"abstract":"<div>Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879319","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

Biochemical characterization of a high affinity phosphate transporter (PiPT) from root endophyte fungus Piriformospora indica 根内生真菌 Piriformospora indica 的高亲和性磷酸盐转运体（PiPT）的生化特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106559

引用次数: 0

Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum 鉴定 Velezensis 杆菌 S161 菌株的几丁质酶及其对数字青霉的抗真菌活性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-31 DOI: 10.1016/j.pep.2024.106562

{"title":"Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum","authors":"","doi":"10.1016/j.pep.2024.106562","DOIUrl":"10.1016/j.pep.2024.106562","url":null,"abstract":"<div>Previous studies have demonstrated the presence of chitinase in Bacillus velezensis through extensive genomic sequencing and experimental analyses. However, the detailed structure, functional roles, and antifungal activity of these chitinases remain poorly characterized. In this study, genomic screening identified three genes—chiA, chiB, and lpmo10—associated with chitinase degradation in B. velezensis S161. These genes encode chitinases ChiA and ChiB, and lytic polysaccharide monooxygenase LPMO10. Both ChiA and ChiB contain two CBM50 binding domains and one catalytic domain, whereas LPMO10 includes a signal peptide and a single catalytic domain. The chitinases ChiA, its truncated variant ChiA2, and ChiB were heterologously expressed in Escherichia coli. The purified enzymes efficiently degraded colloidal chitin and inhibited the spore germination of Penicillium digitatum. Notably, even after losing one CBM50 domain, the resultant enzyme, consisting of the remaining CBM50 domain and the catalytic domain, maintained its colloidal chitin hydrolysis and antifungal activity, indicating commendable stability. These results underscore the role of B. velezensis chitinases in suppressing plant pathogenic fungi and provide a solid foundation for developing and applying chitinase-based biocontrol strategies.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879318","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