Protein and Peptide Letters最新文献

{"title":"Pro-fertility and <i>Antioxidant Potentials</i> of <i>Vigna unguiculata</i> (Cowpea) Protein Isolate and Essential Oil: An <i>In vivo</i> and <i>In silico</i> Studies.","authors":"Olugbenga Samuel Oladimeji, Olasunkanmi Kayode Awote, Nzubechi Olympian Elum","doi":"10.2174/0109298665358634241217094220","DOIUrl":"10.2174/0109298665358634241217094220","url":null,"abstract":"<p><strong>Introduction: </strong><i>Vigna unguiculata</i> (Cowpea), a legume rich in phytochemicals, has been traditionally used to improve fertility and treat various ailments. This study used <i>in-silico</i> and <i>invivo</i> methods to evaluate the effects of cowpea protein isolate and essential oil on reproductive hormonal and antioxidant indices.</p><p><strong>Methods: </strong>Forty (40) female rats were divided into eight groups (n=5). After 14 days of treatment, hormone levels (progesterone, prolactin, testosterone and estradiol) and antioxidant activities (superoxide dismutase (SOD), catalase (CAT) were assessed using biochemical kits and standard procedures. Molecular docking studies were performed using PyRx and Biovia Discovery Studio 2021. The ligands generated through gas chromatography-mass spectroscopy (GCMS) analysis of cowpea oil and the target proteins (SOD and CAT) were from downloaded PubChem and RCSB Protein Data Bank, respectively.</p><p><strong>Results: </strong>The results of this study showed that cowpea essential oil and protein isolate significantly (p<0.05) reduced plasma CAT and SOD activities while increasing their activities in the ovary and liver tissues compared to the infertile untreated group. Consistent administration of either cowpea oil or protein isolate was observed to positively regulate the hormonal indices in the infertile treated groups. Phthalic acid, 2-cyclohexyl ethyl isobutyl ester demonstrated a strong binding affinity and binding constant with SOD and CAT, which suggests that the ligands from cowpea essential oil may have antioxidant and pro-fertility properties that could be developed to treat fertility- related issues.</p><p><strong>Conclusion: </strong>Based on the results of this study, it can be concluded that <i>V. unguiculata</i> has antioxidant property, and can promote fertility, possibly through its rich embedded phytochemicals, which substantiates its traditional claim.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"111-123"},"PeriodicalIF":1.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142953979","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}

{"title":"Recombinant Proteins: Evolution to their Therapeutic Potential.","authors":"Kalyani R Thombre, Krishna R Gupta, Tejaswini P Masne, Milind Janrao Umekar","doi":"10.2174/0109298665387985250710041016","DOIUrl":"10.2174/0109298665387985250710041016","url":null,"abstract":"<p><p>Recombinant proteins, which are produced using recombinant DNA technology, have transformed the domains of biotechnology and biomedicine by allowing the production of proteins that are often expensive or difficult to obtain from natural sources. More than 130 recombinant proteins are currently in clinical use by the US FDA, demonstrating the importance of these proteins in both research and therapeutic applications. Bacterial, yeast, mammalian cell cultures, and hybridoma technology are examples of recombinant protein production systems that have enabled the large-scale production of therapeutic proteins, including monoclonal antibodies, which are now essential tools in disease treatment. From their origins with human insulin in the 1980s to the most recent developments in third-generation proteins, this brief review examines the development of recombinant protein therapies. The first generation concentrated on natural structures; the second generation focused on enhancing safety, pharmacokinetics, and specificity; and the third generation is ready to present innovative formulations and delivery systems. This review also covers the use of recombinant proteins in cancer treatment, different protein production systems, and design techniques that keep improving the safety and effectiveness profiles of protein therapies.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"557-574"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144785124","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}

{"title":"Holographic Proteomics: A Review of Digital Holographic Microscopy Applications in Spatial Proteomics.","authors":"Feng Zhu, Qingwen Wang, Xuejia Zheng, Ruiyuan Chen, Chengcheng Liu, Liu Xiang, Jingquan He, Yong Dai","doi":"10.2174/0109298665404512251021101809","DOIUrl":"10.2174/0109298665404512251021101809","url":null,"abstract":"<p><p>Holoproteomics is a state-of-the-art advancement in spatial proteomics, enabling comprehensive spatial analysis of proteins in tissue microenvironments by combining Digital Holographic Microscopy (DHM) imaging and high-precision laser capture microdissection (LCM) techniques. DHM is an advanced technology that utilizes optical interference principles for non-invasive imaging, providing high-resolution three-dimensional visualization of cells and macromolecules without requiring markers. In proteomics, DHM provides crucial technical support for investigating protein interactions and enables high-precision tracking and analysis of dynamic protein changes. In this review, we systematically survey peer-reviewed literature published in the past five years, with a focus on experimental and clinical studies applying DHM to proteomic analyses. Based on the significant advantages of this technology, we introduce the concept of \"Holographic proteomics\" as an emerging research field with promising future directions.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"693-710"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145524182","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}

{"title":"A Preliminary Study on the Antibacterial Activity of the Secretion of the Levantine Water Frog, <i>Pelophylax bedriagae</i> (Camerano, 1882) (Anura:Ranidae).","authors":"Nazlı Atci, Ebru Tanriverdio, Dincer Ayaz","doi":"10.2174/0109298665385364250917100034","DOIUrl":"10.2174/0109298665385364250917100034","url":null,"abstract":"<p><strong>Introduction: </strong>The skin of amphibians performs some vital roles, such as camouflage, ion and water transport, and gas exchange. Additionally, it plays a significant role in the immune system by preventing pathogen invasion. The secretions produced by the granular glands in the skin possess antimicrobial properties, which help prevent harmful microorganisms from entering the animal's body. The study aims to determine the total protein amounts in the secretion of Pelophylax bedriagae (Levant water frog) distributed in Türkiye and to reveal whether it has antimicrobial properties. In this context, it is a pioneering study on antimicrobial peptides in the skin secretion of Pelophylax.</p><p><strong>Methods: </strong>Field research was carried out at İzmir, Türkiye's Homeros Valley. Because AMPs are species-specific, not sex-specific peptides, both male and female adults were used, regardless of sex. The average weight of the animals used in the experiment was 21 grams. Granular glands were stimulated using a specialized device, and the secretions were collected for analysis. The collected secretions were lyophilized after centrifugation. Then, the total protein amount in the secretion was determined by Bicinchoninic Acid (BCA). The antibacterial activities of the skin secretions against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> were assessed using a plate well diffusion assay. The peptide profiles in the skin secretions were determined using the Tricine-SDS-- PAGE electrophoresis method.</p><p><strong>Results: </strong>The plate well diffusion assay demonstrated that the skin secretions created a 21 mm inhibition zone against E. coli and a 20 mm inhibition zone against S. aureus. The results of the Tricine-SDS-PAGE electrophoresis revealed the presence of peptides with five different molecular weights, including one smaller than 5 kDa. As a result of the analysis, it was determined that P. bedriagae secretion exhibits antimicrobial properties, and many proteins with different molecular masses were identified in the skin secretion.</p><p><strong>Discussion: </strong>There are no skin secretion studies on <i>P. bedriage</i> in the literature. However, activity studies were also conducted on the skin secretion of another Pelophylax species, P. ridibundus. The study examined the antibacterial activities of the skin secretion against some gram-positive and gram-negative bacteria using a plate well diffusion assay. Their study showed that E. coli formed a 21 mm zone and S. aureus formed a 24 mm zone.</p><p><strong>Conclusion: </strong>This study is preliminary, and in future studies, AMPs in skin secretions can be isolated by chromatographic methods, such as HPLC, and peptides can be sequenced and identified in detail.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"650-656"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145233265","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}

{"title":"Optimizing Sleep in Athletes: The Potential of α-Lactalbumin in Nutrition Intervention.","authors":"Jingjing Li, Xuepeng Bian","doi":"10.2174/0109298665363873250623103811","DOIUrl":"10.2174/0109298665363873250623103811","url":null,"abstract":"<p><p>Athletes frequently encounter sleep deprivation due to the demands of high-intensity training and competition, which can significantly impair their physical recovery and athletic performance. α-Lactalbumin (α-LA), a key component of whey protein that is rich in tryptophan, has been shown to promote the synthesis of serotonin and melatonin, thereby regulating sleep cycles. Moreover, α-LA has demonstrated the ability to reduce inflammation and oxidative stress associated with fatigue and stress, further contributing to improved sleep quality. This review provides a critical evaluation of the current evidence supporting the role of α-LA in enhancing sleep quality in athletes through mechanisms such as neurotransmitter regulation, immune function improvement, and enhancement of antioxidant defenses. Additionally, it highlights the necessity for further research into the differential effects of α -LA on sleep across various sports and gender groups, as well as its potential synergistic interactions with other nutrients. These insights are essential for developing optimized nutritional interventions aimed at enhancing athletic performance.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"402-413"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12678987/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144637802","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}

{"title":"Inhibition of Canonical Wnt Signaling in Renal Cell Carcinoma Bone Metastasis: An Immunohistochemical Analysis of DKK1 and LRP5 Expression.","authors":"Zixiong Huang, Yiqing Du, Huaqi Yin, Gongwei Wang, Tao Xu","doi":"10.2174/0109298665357331250416081850","DOIUrl":"10.2174/0109298665357331250416081850","url":null,"abstract":"<p><strong>Background and objective: </strong>Canonical Wnt (Wnt/β-catenin) signaling maintains bone homeostasis by promoting osteoblastic activities. The inhibitory factor, Dickkopf-1 (DKK1), enhances bone resorption in malignant diseases. Low-density lipoprotein-related protein (LRP) 5 is antagonized by DKK1. This study aimed to investigate the expression of DKK1 and LRP5 in renal cell carcinoma bone metastasis (RCC-BM).</p><p><strong>Methods: </strong>RCC-BM patients with paired samples of primary and metastatic lesions were selected for the study (RCC-BM group). RCC patients without any metastasis served as the control group (RCC-only group). Immunohistochemical staining with monoclonal anti-DKK1 and polyclonal anti- LRP5 antibody was conducted on paraffin-embedded slides. The staining results were recorded using scoring according to staining intensity in the renal tissue adjacent to the tumor, primary RCC lesions, and RCC-BM lesions.</p><p><strong>Results: </strong>DKK1 was differently expressed among normal renal tissues, primary RCC, and RCC- BM tissues (p<0.001). The DKK1 expression in primary RCC was significantly lower than that in normal renal tissues (p<0.001) without a difference between the RCC-BM and RCC-only groups. DKK1 expression in bone metastasis was significantly higher than that in primary tumors (p<0.001). For RCC-BM patients, the expression of LRP5 in the primary tumor was significantly lower than that in adjacent renal tissues (p<0.01). The tendency of lower expression was found in primary RCC from RCC-BM patients compared to RCC without metastasis (p=0.073).</p><p><strong>Conclusion: </strong>A \"rebound\" pattern of DKK1 expression in bone metastasis lesions and the decreasing LRP5 expression in primary lesions of RCC-BM patients suggested that Wnt/β-catenin signaling was inhibited in RCC-BM. The overexpression of DKK1 and reduced expression of LRP5 suggest that these markers may be useful for the early prediction of RCC-BM.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"327-334"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144028841","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}

{"title":"Molecular Cloning and Expression of Cryptocyanin Gene Isolated from an Indian Variety of <i>Scylla olivacea</i>.","authors":"Simran Mann, Chittibabu Shanthi, Manu Asthana","doi":"10.2174/0109298665387863250506105601","DOIUrl":"10.2174/0109298665387863250506105601","url":null,"abstract":"<p><strong>Background: </strong>Molting and reproduction play vital roles in the life cycle of brachyuran crabs, and these two processes are closely interconnected. A key player in the molting cycle is cryptocyanin, which is similar to hemocyanin in sequence, size, and structure. Hemocyanin is a copper-containing oxygen-binding protein, while cryptocyanin is a copper-free protein that lacks oxygen-binding capacity.</p><p><strong>Objective: </strong>The goal of the study was to carry out the isolation, cloning, and expression of the partial cryptocyanin gene from the Indian variety of <i>Scylla olivacea</i>.</p><p><strong>Methods: </strong>The partial cryptocyanin gene was isolated from the hemocytes of the <i>S. olivacea</i> male and female crabs by qPCR for comparative expression analysis of the cryptocyanin gene.</p><p><strong>Results: </strong>We successfully amplified, cloned, and expressed a 519 bp partial cDNA encoding cryptocyanin from the Indian variety of<i> Scylla olivacea</i>, within the pRSET-B vector.</p><p><strong>Discussion: </strong>In this study, we conducted a comprehensive analysis of cryptocyanin expression in male and female crabs during the intermolt stage. Our findings revealed Cq values of 28.97 for males and 33.68 for females, highlighting a significantly lower abundance of cryptocyanin protein in female crabs.</p><p><strong>Conclusion: </strong>Our study showed that crustacean cDNA can be effectively expressed in bacterial vectors, and clones were stable for up to 6 months at -80oC. Real-time data showed a significant difference in cryptocyanin levels between male and female crabs. This finding highlights the need for further research with a larger sample size for better understanding.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"368-375"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143106","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}

{"title":"Unveiling the Potential Role of Hesperetin and Emodin as a Combination Therapy to Inhibit the Pancreatic Cancer Progression against the C-Met Gene.","authors":"Rangaraj Kaviyaprabha, Thandaserry Vasudevan Miji, Puthupparambil Shaji Sreelakshmi, Sridhar Muthusami, Palanisamy Arulselvan, Muruganantham Bharathi","doi":"10.2174/0109298665363165250225100109","DOIUrl":"10.2174/0109298665363165250225100109","url":null,"abstract":"&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Pancreatic adenocarcinoma (PAAD) is one of the most prevalent cancers, and it has high death rates. Only 10% of PAAD patients can survive until 5 years. Hence, the improvement of survival rate of the patients should be improved.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Aim: &lt;/strong&gt;The present study used a computational approach to identify novel biomarkers and potentially effective small drug-like molecules in PAAD.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Objective: &lt;/strong&gt;The objective of this study was to identify the Differentially Expressed Genes (DEGs) and survival rate affecting genes (SDEGs) to single out the specific gene responsible for pancreatic cancer and predict the efficacy of interactions with hesperetin and emodin. Further, another objective was to validate the predicted efficacies using an MTT assay.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;The GEPIA2 database was used to analyze the TCGA-PAAD dataset and identify DEGs and SDEGs. Venn identified the commonly scattered genes between the DEGs and SDEGs. Network Analyst v3.0, CytoScape v3.10.1, and cytoHubbawere used to construct protein-protein interactions (PPI) network and identifying hub genes which were described as target proteins. The Protein Data Bank (PDB) and PubChem were utilized to obtain the PDB structure of the target proteins and 13 phytocompounds in SDF format. Molecular docking studies were carried out and visualized by utilizing Autodock vina and Discovery Studio Visualizer v19.1.0.1828. The cytotoxicity was measured in the MiaPaCa-2 cell line after being treated with hesperetin and emodin.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;A total of 9219 Differentially Expressed Genes (DEGs) from the TCGA-PAAD dataset were identified. Among them, 8740 and 479 genes were up and down-regulated with the statistical significance of P ≤ 0.05, respectively. Likely, 500 most survival rate affecting genes (SDEGs) in PAAD patients with a statistical significance of P ≤ 0.05 were identified. The common 137 genes were identified between these obtained DEGs and SDEGs. The survival heat map was delineated for the predicted 137 common genes. Ninety-six genes were identified as the most hazardous genes (highlighted in red). After that, the network was constructed by using PPI for the most hazardous 96 genes. From the constructed PPI network, the highly interacted top 10 genes were identified. The survival analysis was carried out to identify the most hazardous genes and revealed that all the identified genes significantly reduced the survival rate of the patients affected by PAAD. From that, high survival affecting 5 genes, such as CDK1, CENPE, NCAPG, KIF20A, and c-MET, were selected for further analysis. The molecular docking studies were carried out for the identified top 5 genes, with the 13 phytocompounds reviewed previously for anti-- cancer activity. The molecular docking analysis revealed that the hesperetin (binding affinity (BA) = -8.0 kcal/mol; Root mean square deviation (RMSD) = 2.012 Å) and emodin (BA = -8.6 k","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"280-298"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143701385","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}

{"title":"miR-584-5p Regulates MSMO1 to Modulate the AKT/PI3K Pathway and Inhibit Breast Cancer Progression.","authors":"Xin Li, Jie Liu, Lili He, Mi Tian, Yingying Xu, Bing Peng","doi":"10.2174/0109298665339026250114070523","DOIUrl":"10.2174/0109298665339026250114070523","url":null,"abstract":"<p><strong>Introduction: </strong>Endogenous microRNAs (miRNAs) are critical regulators of tumor progression, making their role in breast cancer an important area of investigation.</p><p><strong>Methods: </strong>This study examined the regulation of MSMO1 by miR-584-5p in breast cancer cells. Using bioinformatics and Western blotting, we confirmed MSMO1 expression in breast cancer cells and evaluated its effects on cell migration, invasion, and the AKT signaling pathway. In vivo experiments further supported these findings. The interaction between miR-584-5p and MSMO1 was validated through luciferase reporter assays, while functional studies highlighted the impact of miR-584-5p on cancer progression.</p><p><strong>Results: </strong>Our findings revealed that MSMO1 is upregulated in breast cancer, enhancing cell migration and invasion. Silencing MSMO1 diminished AKT pathway activity, and luciferase assays confirmed MSMO1 as a direct target of miR-584-5p.</p><p><strong>Conclusion: </strong>Overexpression of miR-584-5p suppressed migration and invasion of breast cancer cells. In summary, miR-584-5p is likely to modulate MSMO1 and subsequently regulate the AKT/ PI3K pathway, presenting a promising therapeutic target for breast cancer treatment.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"171-182"},"PeriodicalIF":1.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143415087","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}

{"title":"Design, Expression, and Purification of a Fusion Enzyme Containing Terminal Deoxynucleotidyl Transferase from <i>B. bovis</i> and DNA-Binding Proteins from <i>E. coli</i>.","authors":"Antos Sachanka, Veronika Shchur, Yaraslau Dzichenka, Aleksei Yantsevich","doi":"10.2174/0109298665372636250504084653","DOIUrl":"10.2174/0109298665372636250504084653","url":null,"abstract":"<p><strong>Background: </strong>Gene fusion techniques have yielded promising results in the fusion of thermostable polymerases (Taq and Pfu) with single-stranded and double-stranded DNA-binding proteins. Constructing a terminal deoxynucleotidyl transferase (TdT) fusion enzyme with DNAbinding protein domains can enhance thermostability and broaden the enzyme's application field. This makes it a promising candidate for cost-effective <i>de novo</i> DNA synthesis and a more effective tool for demonstrating apoptosis and detecting viral DNA/RNA.</p><p><strong>Methods: </strong>The design of fusion proteins was based on molecular dynamics and homology modeling. Native and fusion proteins were isolated using affinity chromatography on HisTrap HP. Thermostability was assessed through differential scanning fluorimetry and dynamic light scattering. HPLC analysis was conducted to evaluate enzyme activity.</p><p><strong>Results: </strong>According to the <i>in silico</i> predictions of the fusion protein structure, a homotetramer was formed. The expressed fusion proteins were successfully purified under native conditions, similar to TdT. The total yields of the studied proteins were 130 mg/L for single-stranded binding protein from <i>E. coli</i> (EcSSB), 5 mg/L for TdT, 9 mg/L for TdT_L1_EcSSB, and 7 mg/L for TdT_L2_EcSSB. The measured radius of TdT (3.5 nm) was found to be consistent with a monomeric structure; however, the fusion proteins were expected to form a homotetramer. Additionally, fusion with EcSSB was found to prevent aggregation, which positively affected the thermal stability of the fusion protein. Instead of elongating the substrate by adding nucleotides, the fusion enzyme removed a nucleotide, specifically TTP, from the 3'-end of the DNA strand.</p><p><strong>Conclusion: </strong>The fusion of TdT with EcSSB resulted in increased thermal stability and a reduced ability to add nucleotides to the substrate.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":" ","pages":"353-367"},"PeriodicalIF":1.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144142991","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}