Journal of biotechnology最新文献

{"title":"Development of a DNA endonuclease I-SceI-based scarless genome editing system for Cupriavidus necator","authors":"Suk-Jin Oh ,&nbsp;Gaeun Lim ,&nbsp;Yebin Han ,&nbsp;Heetaek Kim ,&nbsp;Yun-Gon Kim ,&nbsp;Shashi Kant Bhatia ,&nbsp;Yung-Hun Yang","doi":"10.1016/j.jbiotec.2025.07.020","DOIUrl":"10.1016/j.jbiotec.2025.07.020","url":null,"abstract":"<div><div><em>Cupriavidus necator</em> is a promising microbial chassis capable of fixing CO₂ and producing high polyhydroxyalkanoate yields. Consequently, various genetic engineering methods have been explored. While <em>sacB</em>-based homologous recombination (HR) and CRISPR-Cas9 have shown both advantages and disadvantages in <em>C. necator</em>, alternative tools, including the DNA endonuclease <em>I-SceI</em>-mediated HR system could enable precise, scarless genome editing without requiring a large database. We developed a two-plasmid-based <em>I-SceI</em> HR system for efficient gene deletion and insertion in <em>C. necator</em> by altering origin replication and induction systems. The pOUO-1 plasmid was designed for conjugation-based genome integration via first HR, whereas the pOH-4 plasmid was constructed to express <em>I-SceI</em>, inducing second HR. Unlike conventional <em>I-SceI</em> expression strategies, which fail to trigger second HR in <em>C. necator</em>, transformation with pOH-4 alone was sufficient for recombination. A plasmid-curing strategy was optimized to eliminate the highly stable pOH-4 by increasing the incubation temperature to 37°C. Using this optimized system, the <em>phaC</em>₁ gene was successfully knocked out; the <em>phaC</em>_BP-M-CPF4 was inserted at the same site, resulting in a novel poly(3-hydroxybutyrate-<em>co</em>-5-hydroxyvalerate)-producing strain. This newly established <em>I-SceI</em> HR technique significantly simplifies genome engineering in <em>C. necator</em>, reducing the timeframe to a few weeks and facilitating its further applications in synthetic biology.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 285-295"},"PeriodicalIF":3.9,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Numerical simulation analysis of flow field and fabrication of cells-osteochondral scaffold constructs in a spinner flask bioreactor","authors":"Xueyan Hu ,&nbsp;Hezhi Chen ,&nbsp;Hailin Ma ,&nbsp;Jingjing Zhu ,&nbsp;Yuen Yee Cheng ,&nbsp;Haohan Xu ,&nbsp;Kedong Song","doi":"10.1016/j.jbiotec.2025.07.017","DOIUrl":"10.1016/j.jbiotec.2025.07.017","url":null,"abstract":"<div><div>With the vigorous development of bone/cartilage tissue engineering research, the construction system for in vitro preparation of tissue engineered osteochondral repair substitutes is undergoing a transformation from static culture mode to 3D dynamic culture mode. However, for dynamic culture mode, many problems such as the selection of cultivation environment and the optimization of condition parameters need to be solved. In this study, computational fluid dynamics (CFD) was used to simulate and predict the stress conditions of adipose derived stem cells-chitosan₇/gelatin₃/Nano-hydroxyapatite (ADSCs-Cs₇/Gel₃/nHAP) structures and ADSCs-bone-derived scaffold structures, as well as the flow field distribution in spinner flask (SF) at different rotational speeds. Finally, the appropriate operating conditions of SF were optimized. The simulation results showed that SF generated two fluid cycles bounded by the bottom edge of the stirring paddle in the entire fluid flow region, with a fluid circulation region exhibiting a relatively static flow field distribution (compared with the first two cycles) directly below the stirring axis. There was a moderate dynamic pressure and speed under the stirring paddle, making this area the most suitable for fixing the cell-scaffold constructs. Under different rotational speed conditions, the dynamic pressure and fluid shear force of the constructs in SF were positively correlated with the speed. Overall, considering all factors, 50 rpm and 70 rpm were determined as the preferred rotational speed conditions for the ADSCs-Cs₇/Gel₃/nHAP constructs and ADSCs-derived-bone scaffold constructs in SF, respectively. Subsequently, the cell-scaffold complex cultured under SF was implanted at the site of osteochondral defect in New Zealand rabbits, and it was found that new tissues were formed after 4 weeks of culture. These results indicate that SF cultured scaffolds are suitable for repairing rabbit osteochondral defects.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 255-271"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144724530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biostimulant-driven growth enhancement and stress resistance in tomato: The combined impact of alginate, chitosan, and salicylic acid","authors":"Fatima El Amerany ,&nbsp;Meriem Naimi ,&nbsp;Mohammed Rhazi","doi":"10.1016/j.jbiotec.2025.07.022","DOIUrl":"10.1016/j.jbiotec.2025.07.022","url":null,"abstract":"<div><div>Mechanical wounding, a significant cause of yield loss in agricultural crops, has prompted recent efforts to identify effective solutions, such as applying biostimulants that not only improve plant growth but also enhance resistance to mechanical damage. This study evaluates the combined effects of alginate (Al-1, 0.75 mg mL−1), salicylic acid (SA, 100 µM), and chitosan (Ch, 0.75 mg mL−1) on tomato plant growth, biochemical responses, and recovery from mechanical wounding. The results indicate that Al-1 accumulates at the plant cell wall, transitioning from a liquid to a film-like state. During this process, Al-1 also loses over 50 % of its sodium ions and fails to acquire nitrogen ions from Ch. However, the combined application of Al-1, SA, and Ch significantly promotes plant growth and enhances mechanical stress resistance by increasing chlorophyll, sugar, protein, and carotenoid levels, as well as improving xylem development compared to other treatments. Furthermore, the Al-1 +Ch+SA combination elevates H₂O₂ levels and APX activity in adjacent leaves 60 min after wounding; although this response is delayed compared to a individual treatments. These findings suggest that this combination of biostimulants enhances plant resilience to mechanical injury, offering potential for improving crop yield and quality in stress-prone agricultural systems</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 244-254"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144722516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Channel matters: Overcoming diffusion bottlenecks via loop engineering of LinD for enhanced isoprene production","authors":"Julian L. Wissner ,&nbsp;Max-Philipp Fischer ,&nbsp;Wendy Escobedo-Hinojosa ,&nbsp;Jan Klenk ,&nbsp;Bettina M. Nestl ,&nbsp;Jan Seeger ,&nbsp;Anibal Cuetos ,&nbsp;Gideon Grogan ,&nbsp;Javier Iglesias-Fernández ,&nbsp;Sílvia Osuna ,&nbsp;Gloria Saab-Rincón ,&nbsp;Bernhard Hauer","doi":"10.1016/j.jbiotec.2025.07.019","DOIUrl":"10.1016/j.jbiotec.2025.07.019","url":null,"abstract":"<div><div>The selective chemical dehydration leading to C<img>C double bond formation is a challenging reaction that harbors great potential for industrial applications. The cofactor independent bifunctional linalool dehydratase isomerase (LinD) from <em>Castellaniella defragrans</em> catalyzes the reversible dehydration of (<em>S</em>)-linalool to myrcene, as well as its isomerization to geraniol. We previously reported that LinD is able to convert the small alkenol 2-methyl-3-buten-2-ol to the valuable product isoprene. To foster the LinD-catalyzed production of isoprene in a novel recombinant <em>E</em>. <em>coli</em> whole-cell two-phase system, we targeted the active site and a flexible α-helix near the putative substrate channel via enzyme engineering. Interestingly, none of the active site variants exhibited an increased product formation. In contrast, saturation mutagenesis of the 10 amino acids forming the α-helix, identified the variants K103N, R104G, G107T and D112T, which exhibited a 1.73 ± 0.05, 1.56 ± 0.12, 2.08 ± 0.12 and 1.93 ± 0.06-fold increase in product formation compared to the wild-type enzyme, respectively. Notably, a combinatorial approach targeting these four variants led to decreased activity in all cases, compared to the corresponding single-point variants, indicating negative epistatic interactions. Thus, employing the most catalytically efficient single point variant G107T, which exhibited a 28-fold higher <em>k</em>_{cat (app)} compared to the wild-type, a total of 2.8 ± 0.2 mM isoprene was obtained utilizing the whole-cell two-phase system. Crystallographic analysis of G107T revealed only minor structural changes; however, molecular dynamic simulations uncovered striking conformational differences relative to the LinD wild-type, emphasizing the role of altered substrate channel in variant G107T.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 12-21"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731084","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An integrated purification and immobilization strategy for ELP-fusion β-glucosidase by its thermosensitivity and hydrophobicity","authors":"Qing Li ,&nbsp;Jingli Xue ,&nbsp;Xinnan Ma ,&nbsp;Juan Han ,&nbsp;Jiacong Wu ,&nbsp;Xu Bao ,&nbsp;Lei Wang ,&nbsp;Yun Wang","doi":"10.1016/j.jbiotec.2025.07.021","DOIUrl":"10.1016/j.jbiotec.2025.07.021","url":null,"abstract":"<div><div>In the field of biocatalysis, enzymes play a crucial role. However, they are faced with many challenges in practical applications, such as poor operational stability, high production costs and difficulties in recycling. Therefore, the efficient separation, purification and immobilization of enzymes are key to realize their industrial application. In this experiment, Elastin-like polypeptides (ELPs) tags with different sequence lengths, (VPGVG)n = 30, 40, 50, were designed to be fused with β-glucosidase (Glu) for expression. The temperature-responsive properties of ELPs have been utilized to achieve efficient separation and purification of recombinant enzymes, significantly improving the purification efficiency. Furthermore, the immobilization of Glu was achieved by enhancing the adsorption force between the recombinant enzyme and the carrier material based on the hydrophobicity of the ELPs. Compared to the free Glu, the immobilized Glu exhibited excellent thermal stability, pH stability, reusability and storage stability. In the catalytic hydrolysis of carboxymethyl cellulose (CMC), the glycosylation rate was increased by 27.26–28.05 % due to the synergistic action of immobilized Glu and cellulase. Therefore, this study developed an integrated method combining separation, purification with immobilization based on recombinant enzymes with ELPs, aiming to enhance the industrial application of immobilized enzymes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 1-11"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering and laboratory evolution of lactose-utilizing Pseudomonas putida strains","authors":"Signe Saumaa ,&nbsp;Tanel Ilmjärv ,&nbsp;Ingrem Popazova,&nbsp;Lea Ets,&nbsp;Age Brauer,&nbsp;Maia Kivisaar","doi":"10.1016/j.jbiotec.2025.07.015","DOIUrl":"10.1016/j.jbiotec.2025.07.015","url":null,"abstract":"<div><div><em>Pseudomonas putida</em> strain KT2440 has been extensively adopted for bioengineering purposes. Despite of its versatile metabolism, this strain is not able to utilize lactose as a carbon source. In this study, we report the construction of KT2440 derivative that can utilize this disaccharide as a growth substrate. The metabolic engineering of this strain was accompanied with the laboratory evolution of bacteria on lactose minimal media. Specifically, the expression of β-galactosidase gene <em>lacZ</em> on a plasmid was accompanied with integration of galactose Leloir pathway genes <em>galETKM</em> and lactose permease gene <em>lacY</em> into the chromosome of KT2440. Thereafter, the engineered strain KT2440Gal was adapted on minimal medium containing lactose as an only carbon source. Three lactose-utilizing mutants were further characterized for their growth on lactose and other sugars, and mutations required to utilize lactose were confirmed by reverse engineering. This study has expanded a potential use of <em>P. putida</em> KT2440 in bioprocesses that are relayed on utilization of various sugars.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 225-235"},"PeriodicalIF":4.1,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144682641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing sustainable production of high-quality microalgae-derived extracellular vesicles through batch-refeed perfusion cultivation","authors":"Sabrina Picciotto ,&nbsp;Dario Belmonte ,&nbsp;Paola Gargano ,&nbsp;Giorgia Adamo ,&nbsp;Angela Paterna ,&nbsp;Estella Rao ,&nbsp;Thomas Conlon ,&nbsp;Samuele Raccosta ,&nbsp;Daniele Paolo Romancino ,&nbsp;Giulia Smeraldi ,&nbsp;Monica Salamone ,&nbsp;Nicolas Touzet ,&nbsp;Mauro Manno ,&nbsp;Antonella Bongiovanni","doi":"10.1016/j.jbiotec.2025.07.016","DOIUrl":"10.1016/j.jbiotec.2025.07.016","url":null,"abstract":"<div><div>Extracellular vesicles (EVs) are lipid-based nanoparticles with strong potential as therapeutic nanocarriers, but their clinical use is limited by production and cost challenges, especially from human cells. Microalgae-derived EVs (<em>i.e.</em>, nanoalgosomes) offer a sustainable and scalable alternative. In this study, we optimized nanoalgosome production by implementing batch-refeed systems that simulate perfusion conditions to improve microalgal viability by maintaining nutrient levels and reducing toxic metabolites. Compared to standard batch cultures, the batch-refeed strategy yielded ten-fold fewer particles but achieved 1.4-fold higher total EV protein yield, likely reflecting a reduction in non-EV co-isolates in favor of bona fide EVs. This is supported by the results of EV-associated luminal esterase activity and by the increase in membrane-enclosed EVs — detected by fluorescent nanoparticle tracking analysis — in batch-refeed–derived nanoalgosomes compared to standard batch cultures, which suggests that the batch-refeed strategy enhances their functional integrity, possibly by preserving vesicle membrane stability and reducing non-vesicular co-isolates. Furthermore, the batch-refeed strategy achieved a three-fold increase in space-time yield (STY) over conventional batch systems. Nanoalgosomes retained key functional properties post-harvest. In vitro assays confirmed that nanoalgosomes derived from both cultivation methods exhibited similar cytoprotective effects, reducing oxidative stress-induced damage in normal mammary epithelial 1–7 HB2 cells and in MDA-MB-231 breast cancer cells. These findings support the use of microalgae-based perfusion-inspired systems as a green, cost-effective and scalable strategy for producing high-quality EVs for biomedical applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 236-243"},"PeriodicalIF":4.1,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144674891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Untargeted metabolomics profiling reveals carbon source-dependent rhamnolipid congener production in Burkholderia thailandensis E264","authors":"Nadirah Arifin ,&nbsp;Kamalrul Azlan Azizan ,&nbsp;Zainatul 'Asyiqin Samsu","doi":"10.1016/j.jbiotec.2025.07.014","DOIUrl":"10.1016/j.jbiotec.2025.07.014","url":null,"abstract":"<div><div>This study investigated glycerol and oleic acid as carbon sources on RL production and congener diversity in <em>Burkholderia thailandensis</em> E264 (BSL 1), a safer alternative to <em>Pseudomonas aeruginosa,</em> using an untargeted metabolomic approach. <em>B. thailandensis</em> E264 was grown in triplicate for nine days at 30 °C, extracted using ethyl acetate, and analysed using LC/Q-TOF/MS. Results showed 84 RL congeners with different adducts were annotated. Cultures with glycerol primarily produced di-RLs with carbon chain lengths from C_12:2 to C₁₆-C₁₄, whereas cultures with oleic acid produced mono-RLs (C_8:2 to C₁₆-C₁₆). Multivariate analysis of PLS-DA revealed distinct RL profiles in response to different carbon sources, with di-RL-C₁₀-C₁₂ (VIP = 2.15) and mono-RL-C₁₀-C_14:1 (VIP = 1.90) identified as key congeners in the glycerol and oleic acid cultures, respectively. The heatmap highlighted significant fold changes in RL congener abundance (2.94-fold higher di-RL-C₁₄ in glycerol culture and 4.38-fold higher mono-RL-C₈-C₁₀ in oleic acid culture). These findings demonstrate that the carbon source significantly affects RL congener production in <em>B. thailandensis</em> E264, suggesting the potential for RL production optimisation and tailoring congener profiles for specific applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 211-224"},"PeriodicalIF":4.1,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667713","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of a novel virus-like particle platform to the development of Neisseria vaccines","authors":"Laura Burgess Tornaletti ,&nbsp;Aljawharah Fahad Alabbad ,&nbsp;Angela Thistlethwaite ,&nbsp;Smruti Rashmi ,&nbsp;Jeremy P. Derrick","doi":"10.1016/j.jbiotec.2025.07.012","DOIUrl":"10.1016/j.jbiotec.2025.07.012","url":null,"abstract":"<div><div>Development of vaccines against meningococcal meningitis has been driven by the identification of protective protein antigens. In the case of gonorrhoea, this has been less successful, although there is evidence that a vaccine against the causative agent of meningococcal meningitis, <em>Neisseria meningitidis</em>, elicits some protection against infection by <em>Neisseria gonorrhoeae</em>, which causes gonorrhoea. Here we describe an approach that uses antigen fusion to antibody Fc domains and attachment to an engineered virus-like particle (VLP) platform to display multiple <em>Neisseria</em> antigens. Any combination of antigens which are fused to an antibody Fc domain can be attached to the VLP, a technology we have termed ‘AbBind’. We exemplify this invention with seven antigens which are vaccine candidates for protection against meningococcal or gonococcal infection. Antigens are expressed as Fc fusions in HEK cells, purified using Protein A and then added to the AbBind VLP core. Five antigens induce potent IgG antibody responses in mice, as fusions to Fc and/or in complex with the VLP core. This highly adaptable platform integrates well with mammalian expression pipelines which are commonly used for antibody production and is therefore readily scalable. The results raise the prospect of jointly formulated vaccines to elicit protection against both diseases.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 190-197"},"PeriodicalIF":4.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144662494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bak inserts into Mcl-1-puma and Mcl-1-BimL dimers to form hetero-trimers analyzed using quantitative live-cell FRET imaging","authors":"Hongce Chen ,&nbsp;Wenhua Su ,&nbsp;Lingyu Wang ,&nbsp;Zhirui Wu ,&nbsp;Beini Sun ,&nbsp;Xiaoping Wang ,&nbsp;Tongsheng Chen","doi":"10.1016/j.jbiotec.2025.07.013","DOIUrl":"10.1016/j.jbiotec.2025.07.013","url":null,"abstract":"<div><div>This report uses fluorescence resonance energy transfer (FRET) to explore protein-protein interactions and the relative priorities of Bcl-2 family proteins in living cells, focusing on four proteins: Mcl-1, Bak, BimL and Puma. FRET analysis of cells coexpressing CFP-Mcl-1 and YFP-Bak/Puma/BimL demonstrates the direct binding of Mcl-1 to Bak/Puma/BimL. We constructed Bak-P2A-CFP-Mcl-1, BimL-P2A-CFP-Mcl-1, and Puma-P2A-CFP-Mcl-1 plasmids to achieve the co-expression of Bak/Puma/BimL and Mcl-1. FRET analysis for the cells coexpressing Bak-P2A-CFP-Mcl-1 and YFP-Puma/BimL shows that co-expression of Bak and Mcl-1 significantly increased the <em>ED</em>max values between CFP-Mcl-1 and YFP-Puma/BimL, indicating that Bak makes the sites of Mcl-1 and Puma/BimL close by inserting into the Mcl-1-Puma/BimL dimers to form Bak-Mcl-1-Puma/BimL trimers, which has also been further verified by Co-IP analysis. For the cells coexpressing Puma/BimL-P2A-CFP-Mcl-1 and YFP-Bak, the <em>ED</em> values between Mcl-1 and Bak were very small, demonstrating that Mcl-1 binds preferentially to Puma or BimL in the presence of both Puma or BimL and Bak. Collectively, Puma/BimL preferentially binds to Mcl-1 to form Mcl-1-Puma and Mcl-1-BimL dimers, and Bak can insert into the Mcl-1-Puma and Mcl-1-BimL hetero-dimers to form hetero-trimers (Bak-Mcl-1-Puma and Bak-Mcl-1-BimL).</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 168-178"},"PeriodicalIF":4.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144653508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}