Protein and Peptide Letters最新文献

{"title":"A Functional Human Glycogen Debranching Enzyme Encoded by a Synthetic Gene: Its Implications for Glycogen Storage Disease Type III Management.","authors":"Doriana Triggiani, Olivia C Demurtas, Elena Illiano, Silvia Massa, Alessandra Pasquo, Carlo Dionisi-Vici, Carmela Marino, Giovanni Giuliano, Rosella Franconi","doi":"10.2174/0109298665307430240628063339","DOIUrl":"https://doi.org/10.2174/0109298665307430240628063339","url":null,"abstract":"<p><strong>Background: </strong>Glycogen Storage Disease type III (GSD III) is a metabolic disorder resulting from a deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (approximately 170 kDa) with cytoplasmic localization and two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Mutations in the Agl gene, with consequent deficiency in GDE, lead to the accumulation of abnormal/toxic glycogen with shorter chains (phosphorylase limit dextrin, PLD) in skeletal and/or heart muscle and/or in the liver. Currently, there is no targeted therapy, and available treatments are symptomatic, relying on specific diets.</p><p><strong>Methods: </strong>Enzyme Replacement Therapy (ERT) might represent a potential therapeutic strategy for GSD III. Moreover, the single-gene nature of GSD III, the subcellular localization of GDE, and the type of affected tissues represent ideal conditions for exploring gene therapy approaches. Toward this direction, we designed a synthetic, codon-optimized cDNA encoding the human GDE.</p><p><strong>Results: </strong>This gene yielded high amounts of soluble, enzymatically active protein in Escherichia coli. Moreover, when transfected in Human Embryonic Kidney cells (HEK-293), it successfully encoded a functional GDE.</p><p><strong>Conclusion: </strong>These results suggest that our gene or protein might complement the missing function in GSD III patients, opening the door to further exploration of therapeutic approaches for this disease.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141634344","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":"Participation of CWINV and SUS Genes in Sucrose Utilization in the Disruption of Cambium Derivatives Differentiation of Silver Birch.","authors":"Yulia Leonidovna Moshchenskaya, Natalia Alekseevna Galibina, Aleksandra Aleksandrovna Serkova, Tatyana Vladimirovna Tarelkina, Ksenia Michailovna Nikerova, Maksim Anatol'evich Korzhenevsky, Irina Nikolaevna Sofronova, Ludmila Igorevna Semenova","doi":"10.2174/0109298665309207240621094227","DOIUrl":"https://doi.org/10.2174/0109298665309207240621094227","url":null,"abstract":"<p><strong>Background: </strong>The mechanisms that control the accumulation of woody biomass are of great interest to the study. Invertase and sucrose synthase are enzymes that are vital for distributing carbon in various biosynthetic pathways. Karelian birch (Betula pendula var. carelica) is a form of silver birch (B. pendula Roth) and is characterized by disruption of the differentiation of cambium derivatives towards both the xylem and phloem, which leads to a change in the proportion of the conducting tissues' structural elements and the figured wood formation. We researched the expression profiles of genes encoding sucrose-cleaving enzymes (CWINV and SUS gene families) and genes encoding CVIF protein, which is responsible for the post-translational regulation of the cell wall invertase activity.</p><p><strong>Object: </strong>In our study, 16-year-old common silver birch (Betula pendula var. pendula) and Karelian birch were used for sampling non-figured and figured trunk section tissues, respectively. Samples were selected for the research based on the radial vector: non-conductive, conductive phloem, cambial zone - differentiating xylem - mature xylem.</p><p><strong>Method: </strong>The enzyme's activity was investigated by biochemical methods. RT-PCR method was used to determine the level of gene expression. Anatomical and morphological methods were used to determine the stage of differentiation of xylem cambial derivatives.</p><p><strong>Results: </strong>Our research revealed a shift in the composition of xylem components in figured Karelian birch, characterized by increased parenchymatization and reduced vessel quantity. In all studied trunk tissues of Karelian birch, compared with common silver birch, an increase in the expression of the CWINV gene family and the SUS3 gene and a decrease in the expression of SUS4 were shown.</p><p><strong>Conclusion: </strong>Therefore, the increase in parenchymatization in figured Karelian birch is linked to a shift in sucrose metabolism towards the apoplastic pathway, indicated by a higher cell wall invertase activity and gene expression. The expression of the SUS4 gene correlates with the decrease in xylem increments and vessel proportion. The research findings will enhance our understanding of how sucrose breaking enzymes regulate secondary growth in woody plants and aid in developing practical timber cultivation methods.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498758","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":"Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.","authors":"Sara Hemmati, Parvaneh Maghami, Javad Ranjbari, Maryam Tabarzad","doi":"10.2174/0109298665314267240624091046","DOIUrl":"https://doi.org/10.2174/0109298665314267240624091046","url":null,"abstract":"<p><strong>Introduction: </strong>Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1.</p><p><strong>Objectives: </strong>In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli.</p><p><strong>Methods: </strong>The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process.</p><p><strong>Results: </strong>Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity.</p><p><strong>Conclusion: </strong>It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498757","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":"The Multifunction of TRIM26: From Immune Regulation to Oncology.","authors":"Jialai Zou, Kaiyi Niu, Tao Lu, Jianxun Kan, Hao Cheng, Lijian Xu","doi":"10.2174/0109298665311516240621114519","DOIUrl":"https://doi.org/10.2174/0109298665311516240621114519","url":null,"abstract":"<p><p>Ubiquitination, a crucial post-translational modification, plays a role in nearly all physiological processes. Its functional execution depends on a series of catalytic reactions involving numerous proteases. TRIM26, a protein belonging to the TRIM family, exhibits E3 ubiquitin ligase activity because of its RING structural domain, and is present in diverse cell lineages. Over the last few decades, TRIM26 has been documented to engage in numerous physiological and pathological processes as a controller, demonstrating a diverse array of biological roles. Despite the growing research interest in TRIM26, there has been limited attention given to examining the protein's structure and function in existing reviews. This review begins with a concise overview of the composition and positioning of TRIM26 and then proceeds to examine its roles in immune response, viral invasion, and inflammatory processes. Simultaneously, we demonstrate the contribution of TRIM26 to the progression of various diseases, encompassing numerous malignancies and neurologic conditions. Finally, we have investigated the potential areas for future research on TRIM26.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141493179","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":"Circular RNA hsa_circ_0005939 Regulates UHRF1BP1L Expression by Targeting miR-4693-3p to Promote Colorectal Cancer Progression.","authors":"Hua Ge, Yan Yan, Haomin Wang, Jun Bian, Zhilong Deng, Xian Su, Kaiyuan Luo, Jianfeng Bin","doi":"10.2174/0109298665297110240611115010","DOIUrl":"https://doi.org/10.2174/0109298665297110240611115010","url":null,"abstract":"<p><strong>Introduction: </strong>Colorectal cancer (CRC) is the second most common and fatal cancer in China. circRNAs are different expressed between tumor and non-tumor tissues, and they are proved to be correlated with tumorigenesis and cancer progression.</p><p><strong>Objective: </strong>We aimed to explore the biological and molecular function of hsa_circ_0005939 in CRC.</p><p><strong>Methods: </strong>We collected and compared ten CRC tissues and four noncancerous tissues and performed circRNA sequencing. We investigated the hsa_circ_0005939 expression in fresh tissues from CRC and adjacent tissues by qPCR. Meanwhile, functional roles of hsa_circ_0005939 in CRC cells were explored by CCK-8, colony formation, wounding healing, cell apoptosis and western blot assays. RNA-FISH was used to confirm the cellular distribution of hsa_circ_0005939. Bioinformatic prediction and luciferase reporter assay were used to determine the mechanisms of hsa_circ_0005939.</p><p><strong>Results: </strong>Our results indicated that hsa_circ_0005939 was up-regulated in CRC tissues and cells. Up-regulation of hsa_circ_0005939 was associated with the occurrence and the number of lymph node metastasis of CRC. Hsa_circ_0005939 down-regulation inhibited cell proliferation, increased cell apoptosis and caused G2 phase arrest of CRC cells. Mechanistically, luciferase assay revealed that hsa_circ_0005939 acts as a molecular sponge for miR-4693-3p and then enhanced Ubiquitin Like With PHD And Ring Finger Domains 1 binding protein 1 like (UHRF1BP1L) expression.</p><p><strong>Conclusion: </strong>Our findings indicated an oncogenic role of hsa_circ_0005939 in CRC, and it enhanced malignant phenotypes of CRC cells through miR-4693-3p/UHRF1BP1L axis. Our study may offer promising biomarkers and therapeutic targets for CRC.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141451368","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":"Variable Surface Antigens of Plasmodium falciparum: Protein Families with Divergent Roles.","authors":"Jasweer Kaur, Prakash Chandra Mishra, Rachna Hora","doi":"10.2174/0109298665298567240530170924","DOIUrl":"https://doi.org/10.2174/0109298665298567240530170924","url":null,"abstract":"<p><p>Malaria caused by Plasmodium falciparum (Pf) is an illness that contributes significantly to the global health burden. Pf makes significant alterations to the host cell to meet its metabolic demands and escape the immune response of the host. These include the export of a large number of parasite proteins to the infected Red Blood Cells (iRBC). Variable Surface Antigens (VSAs), which are highly polymorphic protein families with important roles in immune evasion, form an important component of the exported proteins. A total of five protein families constitute the VSAs, viz. PfEMP1 (Pf erythrocyte membrane protein 1), RIFIN (repetitive interspersed family), STEVOR (sub-telomeric open reading frame), SURFIN (surface-associated interspersed gene family), and PfMC-2TM (Pf Maurer's cleft two transmembrane). With orthologues present in various simian-infecting species, VSAs take up a variety of domain topologies and organizational structures while exhibiting differential expressions throughout the parasite life cycle. Their expression varies across clinical isolates and laboratory strains, which suggests their crucial role in host cell survival and defense. Members of VSAs are reported to contribute significantly to disease pathogenesis through immune evasion processes like cytoadherence, iRBC sequestration in the host vasculature, rosetting, reduced erythrocyte deformability, and direct immunosuppression. In this study, we have gathered information on various aspects of VSAs, like their orthologues, domain architecture, surface topology, functions and interactions, and three-dimensional structures, while emphasizing discoveries in the field. Considering the vast repertoire of Plasmodial VSAs with new emergent functions, a lot remains unknown about these families and, hence, malaria biology.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141443273","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":"Structural Properties of Rat Intestinal Fatty Acid-Binding Protein with its Dynamics: Insights into Intrinsic Disorder.","authors":"Oyku Irem Balli, Sule Irem Caglayan, Vladimir N Uversky, Orkid Coskuner-Weber","doi":"10.2174/0109298665313811240530055004","DOIUrl":"https://doi.org/10.2174/0109298665313811240530055004","url":null,"abstract":"<p><strong>Background: </strong>The rat intestinal fatty acid-binding protein (I-FABP) is expressed in the small intestine and is involved in the absorption and transport of dietary fatty acids. It is used as a marker for intestinal injury and is associated with various gastrointestinal disorders. I-FABP has been studied extensively using conventional experimental and computational techniques. However, the detection of intrinsically disordered regions requires the application of special sampling molecular dynamics simulations along with certain bioinformatics because conventional computational and experimental studies face challenges in identifying the features of intrinsic disorder.</p><p><strong>Method: </strong>Replica exchange molecular dynamics simulations were conducted along with bioinformatics studies to gain deeper insights into the structural properties of I-FABP. Specifically, the Cα and Hα chemical shift values werecalculated, and the findings were compared to the experiments. Furthermore, secondary and tertiary structure properties were also calculated, and the protein was clustered using k-means clustering. The end-to-end distance and radius of gyration values were reported for the protein in an aqueous solution medium. In addition, its disorder tendency was studied using various bioinformatics tools.</p><p><strong>Results and conclusion: </strong>It was reported that I-FABP is a flexible protein with regions that demonstrate intrinsic disorder characteristics. This flexibility and intrinsic disorder characteristics of I-- FABP may be related to its nature in ligand binding processes.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141443272","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 of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.","authors":"Hui Luo, Yan Zhao, Yuheng Ma, Guodong Liang, Lu Ga, Zhao Meng","doi":"10.2174/0109298665312274240530060233","DOIUrl":"https://doi.org/10.2174/0109298665312274240530060233","url":null,"abstract":"<p><strong>Background: </strong>The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors.</p><p><strong>Objective: </strong>The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction.</p><p><strong>Methods: </strong>The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software.</p><p><strong>Results: </strong>The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation.</p><p><strong>Conclusion: </strong>We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141443271","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":"Biophysical Evidence for the Amyloid Formation of a Recombinant Rab2 Isoform of Leishmania donovani.","authors":"Roshanara, Shivani A. Muthu, Gulafsha, Rati Tandon, A. Selvapandiyan, Basir Ahmad","doi":"10.2174/0109298665299157240327084614","DOIUrl":"https://doi.org/10.2174/0109298665299157240327084614","url":null,"abstract":"BACKGROUND\u0000The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health.\u0000\u0000\u0000OBJECTIVE\u0000This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes.\u0000\u0000\u0000METHODS\u0000The acidic unfolding of rLdRab2 induced at acidic pH is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90⁰ light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy.\u0000\u0000\u0000RESULTS\u0000rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30 % structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils.\u0000\u0000\u0000CONCLUSION\u0000To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140661727","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":"Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.","authors":"M. Karaseva, Vladislav A Gramma, D. Safina, Natalia A Lunina, A. Komissarov, S. Kostrov, I. Demidyuk","doi":"10.2174/0109298665293548240327082821","DOIUrl":"https://doi.org/10.2174/0109298665293548240327082821","url":null,"abstract":"BACKGROUND\u0000Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme.\u0000\u0000\u0000OBJECTIVE\u0000Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of Immobilized Metal Ion Affinity Chromatography (IMAC) for producing 3Cpro.\u0000\u0000\u0000METHODS\u0000We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate.\u0000\u0000\u0000RESULTS\u0000Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence.\u0000\u0000\u0000CONCLUSION\u0000We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140683577","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}