Protein expression and purification最新文献_第9页

Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens 基于重组杆状病毒表达系统生产的 BVDV E2 蛋白作为诊断抗原和免疫原的评估

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-22 DOI: 10.1016/j.pep.2024.106611

Jinke He , Xiaoyu Deng , Xusheng Ma , Liangjia Yao , Yiguo Li , Chuangfu Chen , Yanhua He

{"title":"Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens","authors":"Jinke He , Xiaoyu Deng , Xusheng Ma , Liangjia Yao , Yiguo Li , Chuangfu Chen , Yanhua He","doi":"10.1016/j.pep.2024.106611","DOIUrl":"10.1016/j.pep.2024.106611","url":null,"abstract":"<div><div>Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106611"},"PeriodicalIF":1.4,"publicationDate":"2024-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142314941","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

Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii 植物乳杆菌提取的细菌素对耐多药鲍曼不动杆菌的抗生物膜和抗菌作用

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-19 DOI: 10.1016/j.pep.2024.106610

Kasra Javadi , Mohammad Reza Emadzadeh , Seyed Amir Hossein Mohammadzadeh Hosseini Moghri , Mehrdad Halaji , Hadi Parsian , Mehdi Rajabnia , Abazar Pournajaf

{"title":"Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii","authors":"Kasra Javadi , Mohammad Reza Emadzadeh , Seyed Amir Hossein Mohammadzadeh Hosseini Moghri , Mehrdad Halaji , Hadi Parsian , Mehdi Rajabnia , Abazar Pournajaf","doi":"10.1016/j.pep.2024.106610","DOIUrl":"10.1016/j.pep.2024.106610","url":null,"abstract":"<div><div>This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106610"},"PeriodicalIF":1.4,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142293976","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

Cloning and characterization of a novel nitric oxide synthase gene from Exiguobacterium profundum and its expression in Escherichia coli 一氧化氮合成酶基因的克隆和特性鉴定及其在大肠杆菌中的表达。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-18 DOI: 10.1016/j.pep.2024.106609

Xifan Luo, Xinyu Li, Yaru Zhang, Fei Zhao, Jinlong Wang, Jiang Wu

引用次数: 0

Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus 抗副溶血性弧菌的海洋内溶菌素 LysVPB 的特征

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-16 DOI: 10.1016/j.pep.2024.106608

Juan Chen , Ziyun Zhao , Xiaofeng Mu , Mengxin Wang , Jun Tang , Qingqing Bi

{"title":"Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus","authors":"Juan Chen , Ziyun Zhao , Xiaofeng Mu , Mengxin Wang , Jun Tang , Qingqing Bi","doi":"10.1016/j.pep.2024.106608","DOIUrl":"10.1016/j.pep.2024.106608","url":null,"abstract":"<div>Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0–10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba2+, Co2+, and Cu2+ showed inhibitory effects on the activity of LysVPB, while Ca2+ can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106608"},"PeriodicalIF":1.4,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142239835","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}

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Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops) 对薰衣草扇贝中非特异性脂质转移蛋白 nsLTP 功能机制的认识

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-10 DOI: 10.1016/j.pep.2024.106607

Dafeng Liu , Wenrui Dou , Hongying Song , Huashui Deng , Zhu Tian , Rong Chen , Zhen Liu , Ziwei Jiao , Oren Akhberdi

{"title":"Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops)","authors":"Dafeng Liu , Wenrui Dou , Hongying Song , Huashui Deng , Zhu Tian , Rong Chen , Zhen Liu , Ziwei Jiao , Oren Akhberdi","doi":"10.1016/j.pep.2024.106607","DOIUrl":"10.1016/j.pep.2024.106607","url":null,"abstract":"<div>Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106607"},"PeriodicalIF":1.4,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142164833","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 and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis 来自 Rahnella aquatilis 的新型 L-isoleucine-4-dioxygenase (RaIDO) 的生物化学和结构特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-06 DOI: 10.1016/j.pep.2024.106604

Ruida Shan , Yishu Wang , Shuxin Cheng , Xia Li , Xiaohui Yang , Dengyue Sun , Piwu Li

{"title":"Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis","authors":"Ruida Shan , Yishu Wang , Shuxin Cheng , Xia Li , Xiaohui Yang , Dengyue Sun , Piwu Li","doi":"10.1016/j.pep.2024.106604","DOIUrl":"10.1016/j.pep.2024.106604","url":null,"abstract":"<div>The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106604"},"PeriodicalIF":1.4,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142146096","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 of an effective method for purifying trypsin using a recombinant inhibitor 开发一种利用重组抑制剂纯化胰蛋白酶的有效方法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-02 DOI: 10.1016/j.pep.2024.106597

Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li

{"title":"Development of an effective method for purifying trypsin using a recombinant inhibitor","authors":"Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li","doi":"10.1016/j.pep.2024.106597","DOIUrl":"10.1016/j.pep.2024.106597","url":null,"abstract":"<div>A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106597"},"PeriodicalIF":1.4,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133522","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

Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells 改善中国仓鼠卵巢细胞（CHO）中重组人绒毛膜促性腺激素表达的策略。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-31 DOI: 10.1016/j.pep.2024.106596

Iheb Boukari , Samia Rourou , Dorsaf Bouzazi , Khadija Essafi-Benkhadir , Héla Kallel

{"title":"Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells","authors":"Iheb Boukari , Samia Rourou , Dorsaf Bouzazi , Khadija Essafi-Benkhadir , Héla Kallel","doi":"10.1016/j.pep.2024.106596","DOIUrl":"10.1016/j.pep.2024.106596","url":null,"abstract":"<div>Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 μg/ml) and (14.80 ± 0.13 μg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106596"},"PeriodicalIF":1.4,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142111351","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 simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium 从杂交瘤培养基中制备生物素化免疫球蛋白 M 的简单程序。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-26 DOI: 10.1016/j.pep.2024.106595

Tetsuya Okuda , Katsuya Kato

引用次数: 0

A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation 一种来自隐球菌 WM276 的羧甲基纤维素酶：表达、纯化和表征

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-26 DOI: 10.1016/j.pep.2024.106594

Dylan Moodley, Angela Botes

{"title":"A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation","authors":"Dylan Moodley, Angela Botes","doi":"10.1016/j.pep.2024.106594","DOIUrl":"10.1016/j.pep.2024.106594","url":null,"abstract":"<div>Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some β-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106594"},"PeriodicalIF":1.4,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001669/pdfft?md5=0ce3a90264b1d65ac8b9e512dd0583ef&pid=1-s2.0-S1046592824001669-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142088967","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