Protein expression and purification最新文献_第10页

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome 恶性疟原虫基因组中发现的假定胰岛素酶 PF11_0189 的表达和生化特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-07-01 DOI: 10.1016/j.pep.2024.106539

Prabhash Jyoti Mahanta, Kimjolly Lhouvum

{"title":"Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome","authors":"Prabhash Jyoti Mahanta, Kimjolly Lhouvum","doi":"10.1016/j.pep.2024.106539","DOIUrl":"10.1016/j.pep.2024.106539","url":null,"abstract":"<div>PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 μM and 7.5 μM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 μM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106539"},"PeriodicalIF":1.4,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498763","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 of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-β-1,4-GlcNAc-X 一种新型半乳糖-1-磷酸尿苷酰转移酶的异源表达及其在生物生产 Gal-β-1,4-GlcNAc-X 中的应用。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-06-29 DOI: 10.1016/j.pep.2024.106538

Kaiqi Li

{"title":"Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-β-1,4-GlcNAc-X","authors":"Kaiqi Li","doi":"10.1016/j.pep.2024.106538","DOIUrl":"10.1016/j.pep.2024.106538","url":null,"abstract":"<div>Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and β-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-β-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-β-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106538"},"PeriodicalIF":1.4,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141477280","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

Purification of recombinantly produced somatostatin-28 comparing hydrochloric acid and polyethyleneimine as E. coli extraction aids 使用盐酸和聚乙烯亚胺作为大肠杆菌提取辅助剂纯化重组生产的体生长抑素-28。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-06-27 DOI: 10.1016/j.pep.2024.106537

Matthias Müller , Martin Gibisch , Cécile Brocard , Monika Cserjan-Puschmann , Gerald Striedner , Rainer Hahn

{"title":"Purification of recombinantly produced somatostatin-28 comparing hydrochloric acid and polyethyleneimine as E. coli extraction aids","authors":"Matthias Müller , Martin Gibisch , Cécile Brocard , Monika Cserjan-Puschmann , Gerald Striedner , Rainer Hahn","doi":"10.1016/j.pep.2024.106537","DOIUrl":"10.1016/j.pep.2024.106537","url":null,"abstract":"<div>Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106537"},"PeriodicalIF":1.4,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001098/pdfft?md5=282b26329b15971b5a91ea44106fcd5b&pid=1-s2.0-S1046592824001098-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141470347","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

Isolation, characterization and antimicrobial properties of hepatopancreas lectin of the freshwater crab Oziotelphusa naga 淡水蟹 Oziotelphusa naga 的肝胰腺凝集素的分离、特征和抗菌特性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-06-21 DOI: 10.1016/j.pep.2024.106536

F. Vargila , S. Mary Mettilda Bai , J. Vinoliya Josephine Mary , T. Citarasu

{"title":"Isolation, characterization and antimicrobial properties of hepatopancreas lectin of the freshwater crab Oziotelphusa naga","authors":"F. Vargila , S. Mary Mettilda Bai , J. Vinoliya Josephine Mary , T. Citarasu","doi":"10.1016/j.pep.2024.106536","DOIUrl":"10.1016/j.pep.2024.106536","url":null,"abstract":"<div>Lectins are versatile proteins that specifically recognize and interact with sugar moieties expressed on the cell surface. The potential of lectin in drug targeting and delivery has instigated interest to identify natural lectins. Crabs have been identified as a rich source of lectin because the innate immune system is activated on encounter of pathogens and helps in the production of lectin. Although the presence of lectins in crab's hemolymph is well documented, little information about lectin in hepatopancreas, a vital organ for immunity and digestion in crustaceans, is currently available. A calcium dependent lectin (75 kDa) was purified from the hepatopancreas of the freshwater crab Oziotelphusa naga by bioadsorption and fetuin linked Sepharose 4B affinity chromatography technique. The isolated hepatopancreas lectin is calcium dependent and maximum agglutination was observed with rabbit erythrocytes. The hemagglutinating activity of the hepatopancreas lectin was effectively inhibited by sugars, such as α-lactose, GlcNAc, trehalose and NeuAc. Compared to sialylated N-glycosylated proteins including transferrin and apo transferrin, sialylated O-glycosylated proteins like fetuin exhibited stronger inhibitory effect. The ability of erythrocytes to bind hepatopancreas lectin has been diminished by desialylation of the potent inhibitor, indicating the significance of sialic acid in lectin-ligand interactions. The purified hepatopancreas lectin showed a broad spectrum of antimicrobial activity against bacteria Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, E. coli and fungi Candida albicans and Aspergillus niger. The findings of this study demonstrate the significance of hepatopancreas lectin as a multifunctional defense protein that inhibits the growth of bacteria and fungi.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106536"},"PeriodicalIF":1.4,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141440835","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

Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide 重组人超氧化物歧化酶与resilin-like多肽整合的纯化与特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-06-18 DOI: 10.1016/j.pep.2024.106535

Chengli Zhao , Wenrui Huang , Jiayi Su , Xinshuang Zhang , Jingli Xue , Cailiang Zhang , Juan Han , Yang Zhou , Yun Wang

{"title":"Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide","authors":"Chengli Zhao , Wenrui Huang , Jiayi Su , Xinshuang Zhang , Jingli Xue , Cailiang Zhang , Juan Han , Yang Zhou , Yun Wang","doi":"10.1016/j.pep.2024.106535","DOIUrl":"10.1016/j.pep.2024.106535","url":null,"abstract":"<div>Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106535"},"PeriodicalIF":1.4,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141432651","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 trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative 一种来自 Centrosema plumieri 的新型胰蛋白酶抑制剂，可有效抑制蓖麻毛虫的消化蛋白酶，是一种环保型替代品。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-06-17 DOI: 10.1016/j.pep.2024.106534

Cláudio Wilian Victor dos Santos , Antônio Thomás Da Silva , Andrea Carla de Almeida Barros , Josiel Santos do Nascimento , Luciano Aparecido Meireles Grillo , Francis Soares Gomes , Hugo Juarez Vieira Pereira

{"title":"A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative","authors":"Cláudio Wilian Victor dos Santos , Antônio Thomás Da Silva , Andrea Carla de Almeida Barros , Josiel Santos do Nascimento , Luciano Aparecido Meireles Grillo , Francis Soares Gomes , Hugo Juarez Vieira Pereira","doi":"10.1016/j.pep.2024.106534","DOIUrl":"10.1016/j.pep.2024.106534","url":null,"abstract":"<div>Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106534"},"PeriodicalIF":1.4,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141427455","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 basic biochemical characteristics of recombinant surfactant protein D of bottlenose dolphin (Tursiops truncatus) 瓶鼻海豚（Tursiops truncatus）重组表面活性蛋白 D 的表达和基本生化特征。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-15 DOI: 10.1016/j.pep.2024.106523

Takahisa Hamano , Makio Yanagisawa , Seiji Hobo

引用次数: 0

Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi 蒿属植物中 2,3-氧化喹啉环化酶的分子克隆和功能鉴定。

IF 1.6 4区生物学

Protein expression and purification Pub Date : 2024-06-13 DOI: 10.1016/j.pep.2024.106533

Yaman Chen , Ruoshi Huang , Jiabo Chen , Chumin Lin , Yuhong Wu , Jitong Chen , Qi Shen , Feng Wang , Lixin Duan , Honghua Cui

{"title":"Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi","authors":"Yaman Chen , Ruoshi Huang , Jiabo Chen , Chumin Lin , Yuhong Wu , Jitong Chen , Qi Shen , Feng Wang , Lixin Duan , Honghua Cui","doi":"10.1016/j.pep.2024.106533","DOIUrl":"10.1016/j.pep.2024.106533","url":null,"abstract":"<div>Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, β-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3β-ol, and dammara-20,24-dien-3β-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3β-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and β-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"222 ","pages":"Article 106533"},"PeriodicalIF":1.6,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141321466","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

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>Chlamydia trachomatis (CT) 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 Chlamydia. 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 CT'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 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.</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>The SARS-CoV-2 main protease (Mpro) plays a crucial role in virus amplification and is an ideal target for antiviral drugs. Currently, authentic Mpro is prepared through two rounds of proteolytic cleavage. In this method, Mpro 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 Mpro through single digestion. Mpro 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 Mpro generated by the previous method. These findings indicated that authentic Mpro was successfully obtained. Moreover, the substrate specificity of Mpro was investigated. Mpro 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 Mpro for sudden coronavirus infection in the future.</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