The Protein Journal最新文献

{"title":"Soluble Expression of Recombinant Human Mitsugumin 53 in Escherichia coli NiCo21(DE3)","authors":"Kartika Sari Dewi,&nbsp;Winda Tasia,&nbsp;Dian Fitria Agustiyanti,&nbsp;Hariyatun Hariyatun,&nbsp;Popi Hadi Wisnuwardhani,&nbsp;Yana Rubiyana,&nbsp;Nissa Arifa,&nbsp;Hastuti Handayani Purba,&nbsp;Alfian Mahardika Forentin,&nbsp;Ratna Annisa Utami,&nbsp;Wien Kusharyoto,&nbsp;Andri Wardiana","doi":"10.1007/s10930-026-10319-7","DOIUrl":"10.1007/s10930-026-10319-7","url":null,"abstract":"<div>\u0000 \u0000 <p>Mitsugumin 53 (MG53) shows significant potential as a novel therapeutic agent, offering a variety of benefits, including membrane repairment, antiviral properties, and possible applications in cancer therapy. However, this protein tends to form inclusion bodies when expressed in the bacterial system, despite lacking disulfide bonds. Consequently, multiple stages—such as solubilization-tag removal, protein solubilization, purification, and refolding—are necessary to obtain an active protein, leading to significantly high production costs. In this study, we optimized both the intrinsic and extrinsic factors for the soluble expression of recombinant human MG53 (rhMG53) in <i>Escherichia coli</i> without a solubilization tag, subsequently assessing its wound-healing activity against HaCaT cells. The codon-optimized <i>mg53</i> gene, with 5’ mRNA folding free energy (5-ΔG) higher than − 10 kcal/mol, was cloned into a T7-based expression vector and transformed into <i>E. coli</i> NiCo21(DE3) for protein expression. Several transformants were screened to identify high-expressing colonies. To optimize the expression of rhMG53, we varied the incubation temperature, inducer concentration, and pre-induction growth conditions. The SDS-PAGE results demonstrated that the soluble 53 kDa MG53 protein was successfully expressed under optimal conditions at 25 °C, using 0.5 mM IPTG and induced at an optical density of 0.8. An in vitro scratch assay revealed that the scratch wound was almost completely closed within 24 h with an optimum concentration of 60 µg/mL rhMG53. Techno-Economic Analysis showed that our approach can drastically lower production costs, reducing them by one-fourth compared to the inclusion body approach. The findings highlight the successful soluble expression of MG53 in a bacterial system without the use of a solubilization tag.</p>\u0000 </div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 2","pages":"284 - 297"},"PeriodicalIF":1.4,"publicationDate":"2026-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146151681","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":"Preparation and Evaluation of a Multi-Epitope Peptide for Assessing Immunotherapy of Colorectal Cancer in Vivo","authors":"Fatemeh Bahramibanan,&nbsp;Meysam Soleimani,&nbsp;Amir Taherkhani,&nbsp;Hamidreza Ghadimipour,&nbsp;Rezvan Najafi,&nbsp;Nastaran Barati,&nbsp;Katayoun Derakhshandeh","doi":"10.1007/s10930-026-10322-y","DOIUrl":"10.1007/s10930-026-10322-y","url":null,"abstract":"<div><p>The presence of tumor heterogeneity is a critical issue that restricts the success of targeted therapies and negatively impacts patient outcomes. Recent studies have concentrated on the development of multi-epitope peptides that exhibit considerable overexpression in cancerous tissues with the aim of activating immune cells and utilizing immune-mediated responses to effectively suppress tumor growth. In this study, genes with increased expression in colorectal cancer (CRC) were identified using GEO data and R software. Following overexpression confirmation of APCDD1, we identified epitopes from the protein that can be recognized by various MHC molecules and presented on APC cell surfaces. Subsequent to expression and purification of the multi-epitope peptide and investigation on the BALB/c mice harboring tumor xenograft, obtained results showed a significant reduction in tumor growth, mitotic cell count, angiogenesis, metastasis, and an increase in Tumor-Infiltrating Lymphocytes (TILs) in the tumor microenvironment. Overall, the finding highlight the potential of multi-epitope peptide in CRC immunotherapy, where it may address the significant challenge of tumor heterogeneity.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 2","pages":"298 - 317"},"PeriodicalIF":1.4,"publicationDate":"2026-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146151572","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":"Comment to the Editor: From Pretty Pictures to Decision-Grade Models: A Standards-First Roadmap for cryo-EM × Computation","authors":"M. Vijayasimha","doi":"10.1007/s10930-026-10320-0","DOIUrl":"10.1007/s10930-026-10320-0","url":null,"abstract":"","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 1","pages":"5 - 7"},"PeriodicalIF":1.4,"publicationDate":"2026-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146069480","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":"In Memoriam: Professor Ruth Ann Austad Altschuld (1940–2025)","authors":"Prakash Narayan,&nbsp;Lawrence J. Berliner,&nbsp;Mahesh Narayan","doi":"10.1007/s10930-025-10318-0","DOIUrl":"10.1007/s10930-025-10318-0","url":null,"abstract":"","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 1","pages":"3 - 4"},"PeriodicalIF":1.4,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146000286","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":"Novel Thermostable α-Amylase from Bacillus subtilis: Molecular Characterization, Optimization, and Docking-Based Substrate Profiling","authors":"Shazeen Shoaib,&nbsp;Shumaila Naz,&nbsp;Iram Manzoor,&nbsp;Mahjabeen Saleem,&nbsp;Nadia Zeeshan,&nbsp;Muhammad Sajjad","doi":"10.1007/s10930-025-10315-3","DOIUrl":"10.1007/s10930-025-10315-3","url":null,"abstract":"<div><p>Bacterial alpha-amylases have diverse industrial applications in food, fermentation, and pharmaceuticals. This study focuses on the isolation and characterization of a novel alpha-amylase-producing bacterium through molecular and in silico analyses, including molecular docking to determine enzyme-substrate specificity and binding interactions. Among nine bacterial isolates, S4 demonstrated the highest amylolytic activity of 63.68 U/ml. Molecular identification revealed isolate (S4) identity as <i>Bacillus subtilis</i> (OM278386). Enzyme charcterization revealed that maximum enzyme activity was observed at 40 °C and pH 7.0, after 24 h. The full-length novel alpha-amylase gene from <i>B. subtilis</i> (S4) was amplified, sequenced, and translated into a protein sequence. A putative protein was subjected to BLASTp, phylogenetic analysis, and physicochemical characterization. A 3D model was generated and validated through homology modeling. Molecular docking was performed using six substrates: amylopectin, maltotetraose, glycogen, starch, amylose, and cyclodextrin to determine substrate specificity. The putative AmyE protein comprised 488 amino acids. Phylogenetic analysis confirmed its close association with alpha-amylases of other <i>Bacillus</i> species. The enzymes exhibited industrially desirable traits, including high stability, thermotolerance, and hydrophilicity. In contrast, 3D model investigation showed excellent stereochemical quality, with 95.2% of amino acids in the favored region of the Ramachandran plot. Docking studies revealed the highest affinity for amylopectin (binding energy: – 7.2 kcal/mol). Two essential amino acid residues, Asp and Glu-318, were identified as crucial for active-site substrate interactions and enzyme catalysis across various substrates. In conclusion, the analysis presents alpha-amylase from <i>B. subtilis stain</i> S4 as a promising candidate for diverse industrial applications, offering cost-effective alternatives for starch processing, food preservation, and other biotechnological processes.</p><h3>Graphical abstract</h3><p>Schematic presentation of the study workflow illustrates the screening and isolation of a novel alpha-amylase producing isolate (S4) from soil, followed by molecular identification using 16S rDNA sequence. Full-length novel gene is amplified, sequenced, and translated to protein sequence. The three-dimensional model was validated by homology modelling for subsequent docking and substrate binding affinity analysis. </p><div><figure><div><div><picture><source><img></source></picture><span>The alternative text for this image may have been generated using AI.</span></div></div></figure></div></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 2","pages":"332 - 348"},"PeriodicalIF":1.4,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146000331","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":"Non-specific Protein and Peptide Antibacterial Factors of Mammals","authors":"Pavel Levashov,&nbsp;Ilia Zaitsev,&nbsp;Sergei Zaitsev,&nbsp;Daria Gasanova","doi":"10.1007/s10930-025-10314-4","DOIUrl":"10.1007/s10930-025-10314-4","url":null,"abstract":"<div><p>The review summarises the basic information on non-specific factors of mammals that potentially have antimicrobial action. A comparison of previously known factors with the latest literature data is carried out. The following peptide and protein factors are considered: lysozymes, transferrins, interferons, interleukin-2, antimicrobial peptides (defensins, cathelicidins, histatins) and protective glycoproteins (mucins, lectins). These major antibacterial factors perform regulatory functions in the immune system, and some are also able to resist viral and fungal infections or oncological pathologies. The study of the internal antibacterial factors of mammals and the mechanisms of their activation is of great importance for the fight against bacterial infections, including antibiotic-resistant ones. This knowledge is necessary for the development of new approaches to the treatment of humans and farm animals.</p><h3>Graphical Abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 1","pages":"83 - 99"},"PeriodicalIF":1.4,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146000341","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":"Structure, Mutation, Functional Domain Roles and Medical Implications of Glycerol Kinase","authors":"Emmanuel Oluwadare Balogun,&nbsp;Israel Ogwuche Ogra,&nbsp;Uche Samuel Ndidi,&nbsp;Daniel Thakuma Tizhe,&nbsp;Okechukwu Kalu Iroha,&nbsp;Godwin Unekwuojo Ebiloma,&nbsp;Ghulam Jeelani,&nbsp;Tomoyoshi Nozaki,&nbsp;Jair Siqueira-Neto,&nbsp;Harry P. De Koning,&nbsp;Tomoo Shiba","doi":"10.1007/s10930-025-10316-2","DOIUrl":"10.1007/s10930-025-10316-2","url":null,"abstract":"<div><p>Glycerol kinase (GK) is a key part of glycerol metabolism. It connects the metabolic pathways for lipids and carbohydrates by phosphorylating glycerol to glycerol-3-phosphate in an ATP-dependent reaction. This is essential for maintaining carbohydrate homeostasis, plasma glycerol withdrawal, and the utilization of glycerol by different tissues. Together, these processes impact glucose uptake and lipid metabolism. This review discusses the structure of GK, highlights the implications of mutations in the primary sequence, and provides insights on the roles of the various functional domains in the GK-catalyzed reaction. It also discussed the roles of GK in glycerol metabolism, energy production, and its connections with various cellular pathways and disease conditions. The proper regulation of GK activity is crucial, reflecting its critical role in various important cellular processes. Therefore, its regulation has been analyzed from the gene level to posttranslational modification and has implications for GK-linked disease. Separately, the critical role of this enzyme in some disease-causing organisms made it a promising target for inhibitor development. We here explore the current state of GK inhibitor research and discuss strategies for their development. Challenges in GK inhibitor research are identified, and approaches such as high-throughput screening, structure-based drug design, and computational modelling for discovering novel inhibitors are reviewed. Finally, the review highlights critical areas for further research, including the role of GK in synthetic biology and tumour development, among others.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 1","pages":"55 - 82"},"PeriodicalIF":1.4,"publicationDate":"2026-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986149","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":"SARS-CoV-2 Spike Protein S2 Subunit: Recombinant Protein Expression Analysis, Purification, and Its Regulatory Effect on IGF-1R Expression","authors":"Ekta Singh,&nbsp;Rajnish Kumar,&nbsp;Nishita Nishi,&nbsp;Mudita Tripathi,&nbsp;Rani Kumari,&nbsp;Krishna Prakash","doi":"10.1007/s10930-025-10317-1","DOIUrl":"10.1007/s10930-025-10317-1","url":null,"abstract":"<div><p>SARS-CoV-2 consists of the spike (S) protein which plays an important role in mediating the entry of virus into the host and it mainly consists of two subunits which are functionally different from each other. The first subunit S1, is involved in binding to the host receptor such as ACE2, and the second subunit S2, is involved in facilitating the viral membrane fusion with that of host membrane. Despite extensive research on the S1 domain due to its immunodominance and variability, the structurally conserved S2 domain remains relatively understudied. Recognizing the potential of S2 in modulating host responses, this study focuses on its recombinant expression, purification, and functional impact. The S2 coding region was cloned into the pGEX2TK plasmid to produce protein, which is a GST fusion-based protein and thereafter, the expression was done in BL21(DE3) strain of <i>Escherichia coli</i> cells. To enhance yield as well as solubility, protein expression was induced at a reduced temperature of 16 °C, minimizing aggregation and degradation. The fusion protein was purified via glutathione affinity chromatography, yielding high-purity S2 suitable for downstream applications. The S2 protein upon transfection into HEK293 and WI-38 mammalian cells leads to the expression of downregulated insulin-like growth factor 1 receptor (IGF-1R), as measured with the help of protein analysis. In order to highlight the role in pathogenesis of COVID-19 through modulation of cellular receptor and for the intervention of therapeutics, S2 can likely be considered a potential target.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 2","pages":"400 - 408"},"PeriodicalIF":1.4,"publicationDate":"2026-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986147","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":"Production Enhancement of Surface-Expressed D-hydantoinase and D-carbamoylase in E. Coli by Developing a Novel Coculture Process for the Efficient Biotransformation of D-p-hydroxyphenylglycine","authors":"Fatemeh Poureini,&nbsp;Valiollah Babaeipour,&nbsp;Rasoul Khalilzadeh,&nbsp;Reza Hasan Sajedi","doi":"10.1007/s10930-025-10312-6","DOIUrl":"10.1007/s10930-025-10312-6","url":null,"abstract":"<div><p>D-p-hydroxyphenyl glycine (D-PHPG) is a D-amino acid used as an intermediate in the synthesis of semi-synthetic antibiotics. It is synthesized from hydantoin derivatives through two sequential enzymatic reactions involving D-hydantoinase (D-hase) and D-carbamoylase (D-case). Although whole-cell biocatalysis of D-PHPG is cost-effective, its efficiency suffers from transport obstacles, intracellular degradation, and limited substrate solubility. This study utilized a bacterial surface display system to express D-hase and D-case in <i>Escherichia coli</i> for D-PHPG production. Enzyme production optimization was carried out in two stages. Initially, key factors influencing cell density during co-culture were identified through culture media and fermentation parameters screening using the Plackett-Burman design, followed by optimization with the D-optimal method. Next, induction parameters were fine-tuned using response surface methodology. The optimal culture medium was found to contain glycerol (12 g/L) and yeast extract (15 g/L) under optimal induction conditions (0.17 mM IPTG, OD₆₀₀ of 1.3, and 21 °C). These conditions achieved an OD₆₀₀ of 15.6, with expression levels of 20.18% for D-case and 20.82% for D-hase. Scaling up in a stirred tank bioreactor resulted in an OD₆₀₀ of 32.15, with D-hase and D-case expression levels increasing to 25.8 and 24.2%, respectively, and enzymatic activities improving by 2.83 times for D-case and 3.42 times for D-hase. The optimized co-culture approach under optimized induction conditions achieved a conversion yield of 95% and a D-PHPG production yield of 90%. The study results showed that the suggested fermentation conditions will contribute to future scale-up studies aimed at improving enzyme activities for other surface protein production.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 2","pages":"264 - 283"},"PeriodicalIF":1.4,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145859725","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 Characterization of an Engineered CD80 Variant for CTLA-4 Blockade in Cancer Immunotherapy","authors":"Zahra Hajihassan,&nbsp;Amir Hossein Mosayebi,&nbsp;Ramezan Ali Taheri,&nbsp;Parisa Bazargannia","doi":"10.1007/s10930-025-10313-5","DOIUrl":"10.1007/s10930-025-10313-5","url":null,"abstract":"<div>\u0000 \u0000 <p>Immune checkpoint inhibitors, such as monoclonal antibodies or engineered small molecules, are a novel approach to cancer immunotherapy and are being used to treat several types of cancer. CTLA-4, an immune checkpoint, is expressed on the surface of activated T cells where it downregulates immune responses. CD80, also known as B7-1, is a common ligand for CTLA-4 and the T cell co-stimulatory protein CD28. In this study, to inhibit CTLA-4, a variant of the extracellular domain of CD80 (mCD80) carrying the R29Y, Y31R, and Q33K mutations to increase its binding affinity to CTLA-4 was recombinantly expressed in <i>Escherichia coli</i>. The equilibrium dissociation constant (K_D) for the binding of mCD80 to both CD28 and CTLA-4 was also calculated using surface plasmon resonance (SPR) technology. The results showed a significant increase in the binding affinity of mCD80 to CTLA-4, with a 69.76-fold increase compared to wild-type CD80. In contrast, the binding affinity of mCD80 to CD28 showed a significant decrease compared to its wild-type counterpart. These findings suggest that mCD80 may serve as a promising candidate for inhibiting the CTLA-4 on the surface of T cells. Consequently, it may be developed in the future as a potential pharmaceutical agent for cancer immunotherapy.</p>\u0000 </div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":"45 2","pages":"254 - 263"},"PeriodicalIF":1.4,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145859638","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}