Engineering in Life Sciences最新文献

{"title":"Process Intensification for Recombinant Marburg Virus Glycoprotein Production Using Drosophila S2 Cells","authors":"Sven Göbel,&nbsp;Ludwig Mayerlen,&nbsp;Isabelle Yazel Eiser,&nbsp;Lisa Fichtmueller,&nbsp;David Clements,&nbsp;Udo Reichl,&nbsp;Yvonne Genzel,&nbsp;AxelT. Lehrer","doi":"10.1002/elsc.70022","DOIUrl":"https://doi.org/10.1002/elsc.70022","url":null,"abstract":"<p><i>Marburg marburgvirus</i> (MARV) is a highly virulent human pathogen with limited therapeutic options. Recombinant MARV glycoprotein (GP) produced in <i>Drosophila</i> Schneider 2 (S2) cells has been extensively investigated as potential vaccine antigen with promising efficacy demonstrated in nonhuman primate models. However, the existing production process for MARV-GP involving static batch cell cultures with limited scalability and process control show lower than desirable yields. Here, we assessed various process intensification strategies in single-use orbital shaken bioreactors (OSBs) or rocking bioreactors (WAVE) and report maximum viable cell concentrations (VCCs) of 31.6 × 10⁶ cells/mL in batch, 69.5 × 10⁶ cells/mL in fed-batch (FB), and up to 210.0 × 10⁶ cells/mL in perfusion mode. By changing from a glucose-only feed to a CellBoost5 feed, MARV-GP yields were increased by over two-fold. Implementation of perfusion cultures achieved a peak MARV-GP concentration of 57.4 mg/L and a 540% higher space-time yield compared to the FB process in the 50 L WAVE system. However, maximum cell-specific productivities were achieved at a VCC of 85 × 10⁶ cells/mL and decreased with increasing cell concentrations. Glycoanalysis revealed a uniform paucimannosidic <i>N</i>-glycan profile, predominantly α-1,6-core-fucosylated Man3F (F(6)M3) structures, across all production modes. Notably, transitioning pH control from CO₂ to phosphoric acid shifted glycan profiles toward higher mannose forms, highlighting the influence of culture conditions on glycosylation.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144091423","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":"Influence of Exposure Time and Driving Frequency on Cytotoxicity in In Vitro Ultrasound With Constant Mechanical Indices","authors":"Taigo Oyama,&nbsp;Chikahiro Imashiro,&nbsp;Yuta Kurashina,&nbsp;Keita Ando,&nbsp;Kenjiro Takemura","doi":"10.1002/elsc.70011","DOIUrl":"https://doi.org/10.1002/elsc.70011","url":null,"abstract":"<p>Sonochemistry has become increasingly important in bioengineering research, and many in vitro and in vivo bioapplications have been developed. Cytotoxicity is always a concern in its implementation. For in vivo treatments and studies, mechanical index (MI) is known to ensure biocompatibility, and even in vitro MI has been used. Because cell characteristics and acoustic phenomena differ in vitro and in vivo, we questioned using MI in vitro. The in vitro cytotoxicity of ultrasound exposure should be investigated to support the development of cutting-edge sonochemistry. In this study, a system for irradiating cultured cells with 1–2 MHz-range ultrasound was developed to demonstrate the invalidity of employing MI alone in vitro. The results showed that cell damage is defined by the MI, ultrasound frequency, and exposure time, which are new indices for quantifying cell damage. Furthermore, cavitation and acoustic streaming are shown to be the main scientific factors that injure cells.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143949988","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":"Design of a Biocatalytic Filter for the Degradation of Diclofenac and Its Ozonation Products","authors":"Dorothee Schmiemann,&nbsp;Jessica Schneider,&nbsp;Marcel Remek,&nbsp;Jeremy Kaulertz,&nbsp;Oliver Seifert,&nbsp;Monika Weidmann,&nbsp;Klaus Opwis,&nbsp;Arno Cordes,&nbsp;Martin Jäger,&nbsp;Jochen Stefan Gutmann,&nbsp;Kerstin Hoffmann-Jacobsen","doi":"10.1002/elsc.70024","DOIUrl":"https://doi.org/10.1002/elsc.70024","url":null,"abstract":"<p>Posttreatment of the effluents from wastewater treatment plants is becoming increasingly important, as the conventional treatment cannot completely remove organic trace contaminants. Promising techniques like chemical oxidation methods, including ozonation, face the challenge of potentially generating more toxic transformation products than their parent substances due to incomplete oxidation. In this work, the laccase from <i>Trametes versicolor</i> was immobilized on a polyester textile to create a biocatalytic textile filter for the posttreatment of organic trace contaminants and their ozonation by-products. Different filter designs for reactive filtration with biocatalytic textiles were implemented on the laboratory scale and tested for their effectiveness in degrading the dye Remazol Brilliant Blue, the pharmaceutical diclofenac, and its ozonation products. The plate module, inspired by lamellar clarifiers and featuring the textile with covalently immobilized enzyme on the lamella surfaces, exhibited the best performance characteristics. Employing this module, a continuous process of diclofenac ozonation and subsequent posttreatment with the biocatalytic filter was conducted. This not only demonstrated the feasibility of continuous biocatalytic wastewater filtration but also highlighted improved degradation efficiencies of ozonation products compared to the batch process using laccase in solution.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 5","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143901004","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":"Influenza A Virus Production Following Quality by Design Principles","authors":"Tilia Zinnecker,&nbsp;Kristin Thiele,&nbsp;Timo Schmidberger,&nbsp;Yvonne Genzel,&nbsp;Udo Reichl","doi":"10.1002/elsc.70027","DOIUrl":"https://doi.org/10.1002/elsc.70027","url":null,"abstract":"<p>Establishing manufacturing processes for cell culture-based pharmaceutical products involves managing multiple parameters that can affect yield and efficiency, as well as process robustness and product quality. Implementing Quality by Design (QbD) principles can support process optimization, while streamlining the chemistry, manufacturing, and control aspects for regulatory approval. In this study, we mimic a QbD approach based on an influenza A virus production process using two clonal suspension Madin-Darby canine kidney (MDCK) cell lines with distinct characteristics. We performed a quantitative risk assessment including biological and technical parameters to identify the Critical Process Parameters (CPPs). To comprehensively study the effects and interactions of four CPPs, we used an Ambr 15 scale-down system following a Design of Experiments (DoE) approach. After data analysis and modeling, we obtained design spaces characterized by high robustness with a less than 1% risk of failure and even some indications for virus titer and yield improvement, while keeping process-related impurities such as DNA and total protein concentration low. These findings were subsequently verified at a more than 100-fold higher working volume. Taken together, our approach may stimulate ideas for the implementation of streamlined process development and regulatory approval in the field of viral vaccine production.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 4","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70027","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143861810","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":"Automation of a Capillary-Wave Microbioreactor Platform to Enhance Phage Sensitivity Screen Efficiency","authors":"Kevin Viebrock,&nbsp;Ilka Knoke,&nbsp;Leon Huß,&nbsp;Detlev Rasch,&nbsp;Sven Meinen,&nbsp;Andreas Dietzel,&nbsp;Rainer Krull","doi":"10.1002/elsc.70021","DOIUrl":"https://doi.org/10.1002/elsc.70021","url":null,"abstract":"<p>To increase their throughput, reduce laboratory work and improve reproducibility, automation of bioprocesses is gaining in importance nowadays. This applies in particular to microbioreactors (MBRs), which can be easily integrated in highly parallelized and automated platforms and, therefore, be applied for screenings, cell-based assays, and bioprocess development. One promising pharmaceutical application for MBRs is the performance of phage sensitivity tests called phagograms in phage therapy. However, there is no automated and parallelized platform available so far that fulfills the requirements of phagograms. Therefore, a novel highly parallelizable capillary-wave microbioreactor (cwMBR) with a volume of 7 µL, which has already been successfully applied for phagograms, was extended by an in-house built platform for automated fluid addition in the single-digit nanoliter range. The cwMBR has a phage-repellent hydrophilic glass surface. Furthermore, a custom-made highly parallelizable device for biomass measurement in the lower microliter scale was developed and validated in the cwMBR. To prove the applicability of the platform for the generation of phagograms, a phagogram using <i>Escherichia coli</i> and automated phage addition was performed. The results indicate a clear lysis of the bacteria by the phages and thus confirm the applicability of performing automated phagograms in the highly parallelizable cwMBR platform.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 4","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70021","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143831070","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":"Meet Our Editorial Board—Engineering in Life Sciences. An Interview With Michael Zavrel","authors":"Paul Trevorrow,&nbsp;Michael Zavrel","doi":"10.1002/elsc.70023","DOIUrl":"https://doi.org/10.1002/elsc.70023","url":null,"abstract":"&lt;p&gt;&lt;/p&gt;&lt;p&gt;Professor Zavrel studied Chemical Engineering at TUM with a semester abroad at the University of California in Santa Barbara, USA. After completing his diploma thesis at Roche Diagnostics, he did his doctorate at the Chair of Biochemical Engineering at RWTH Aachen University. From 2008 to 2022, he worked in industrial research and development at Süd-Chemie and Clariant, including as Head of Development &amp; Biomanufacturing and as Site Manager. In 2022, Prof. Zavrel was appointed to the professorship for bioprocess engineering at the TUM.&lt;/p&gt;&lt;p&gt;I am a professor of bioprocess engineering at the Technical University of Munich, specializing in the utilization of renewable resources. My research focuses on developing bioprocesses that use enzymes and microorganisms to convert agricultural residues, such as wheat straw and sugar cane bagasse, into products like biopolymers, biofuels, and bio-based chemicals. Previously, I spent a significant period in industry, working at Süd-Chemie and later at Clariant, where I held several positions, including the head of bioprocess development and biomanufacturing.&lt;/p&gt;&lt;p&gt;Although I did not initially plan to return to academia, an unforeseen opportunity arose, leading me to apply for this new professorship. Starting from scratch, I have been building my team and setting up the necessary equipment. My experience in scaling up processes from laboratory to industrial scale is seldom among those who have remained solely in academia. This expertise allows me to contribute significantly to the academic environment by focusing on technology transfer from the lab to larger scales, including cost calculations and life cycle assessments.&lt;/p&gt;&lt;p&gt;I am honored to be a member of the editorial board and look forward enthusiastically to participating actively in this role. My collaboration with partners, demonstration plants, and pilot plants ensures that my work remains practically oriented, bridging the gap between basic science and large-scale industrial applications.&lt;/p&gt;&lt;p&gt;I aim to contribute to trends such as using renewable materials over fossil-based ones, ensuring these materials don't compete with the food chain. With the growing population and limited agricultural space, it's crucial to utilize all parts of plants efficiently. For example, using lignocellulosic parts for chemical processes.&lt;/p&gt;&lt;p&gt;I'm also concerned about the increasing presence of micro and nano plastics, which recycling cannot fully address. Developing biodegradable biopolymers that do not persist in the environment is essential.&lt;/p&gt;&lt;p&gt;Additionally, I focus on leveraging digitalization and artificial intelligence for better fermentation control through pattern recognition and continuous improvement.&lt;/p&gt;&lt;p&gt;That's a good question. I have a family with two young children, aged eight and eleven, which occupies most of my personal time. I greatly enjoy spending time with them. Additionally, I engage in running and hiking, activities that my childre","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 4","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70023","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143831072","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":"Designing Smartly: Understanding the Crystallinity of Melt Electrowritten Scaffolds","authors":"Piotr Stanisław Zieliński,&nbsp;Zhaohang Zhang,&nbsp;Ilaria Squillante,&nbsp;Guillermo Monreal Santiago,&nbsp;Marcus Koch,&nbsp;Giuseppe Portale,&nbsp;Marleen Kamperman,&nbsp;Anastasiia Krushynska,&nbsp;Małgorzata Katarzyna Włodarczyk-Biegun","doi":"10.1002/elsc.70020","DOIUrl":"https://doi.org/10.1002/elsc.70020","url":null,"abstract":"<p>Melt Electrowriting (MEW) is a powerful technique in tissue engineering, enabling the precise fabrication of scaffolds with complex geometries. One of the most important parameters of MEW is collector speed, which has been extensively studied in relation to critical translation speed. However, its influence on crystallinity was overlooked. Crystallinity is crucial for the mechanical properties and degradation behavior of the scaffolds. Therefore, in this study, we present how printing affects the crystallinity of fibers and the resulting mechanical properties of MEW scaffolds. In systematic analysis, we observed a significant reduction in scaffold crystallinity with increased speed, as evidenced by wide-angle X-ray scattering. This decrease in crystallinity was attributed to differences in cooling rates, impacting the polycaprolactone molecular orientation within the fibers. By using tensile testing, we observed the decrease in scaffold Young's modulus with increasing collector speed. Given the relation between crystallinity and mechanical properties of the material, we developed a finite element analysis model that accounts for changes in crystallinity by employing distinct bulk Young's modulus values to help characterize scaffold mechanical behavior under tensile loading. The model reveals insights into scaffold stiffness variation with different architectural designs. These insights offer valuable guidance for optimizing 3D printing to obtain scaffolds with desired mechanical properties.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 4","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143831069","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":"Engineering Caffeic Acid O-Methyltransferase for Efficient De Novo Ferulic Acid Synthesis","authors":"Huai Qi Shang,&nbsp;Qing Bo Yang,&nbsp;Shan Qiang,&nbsp;Rong Zheng,&nbsp;Chao Qun Zhang,&nbsp;Ching Yuan Hu,&nbsp;Qi Hang Chen,&nbsp;Yong Hong Meng","doi":"10.1002/elsc.70018","DOIUrl":"https://doi.org/10.1002/elsc.70018","url":null,"abstract":"<p>Ferulic acid is a high-value chemical synthesized in plants. The ferulic acid biosynthesis is still affected by the insufficient methylation activity of caffeic acid O-methyltransferase (<i>COMT</i>). In this study, we engineered <i>COMT</i> from <i>Arabidopsis thaliana</i> to match caffeic acid, and the mutant <i>COMT</i>^{N129V-H313A-F174L} showed 4.19-fold enhanced catalytic efficiency for degrading caffeic acid. Then, we constructed the de novo synthesis pathway of ferulic acid by introducing tyrosine ammonia lyase from <i>Flavobacterium johnsoniae</i> (<i>FjTAL</i>), 4-hydroxyphenylacetate 3-hydroxylase from <i>Escherichia coli</i> (<i>EcHpaBC</i>), and mutant <i>COMT</i>^{N129V-H313A-F174L}, and further increased tyrosine synthesis. Furthermore, we overexpressed two copies of <i>COMT</i>^{N129V-H313A-F174L} and enhanced the supply of S-adenosyl-L-methionine (SAM) by expressed S-ribosylhomocysteine lyase (<i>luxS</i>) and 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (<i>mtn</i>) to increase the production of ferulic acid. Finally, the production of ferulic acid reached 1260.37 mg/L in the shake-flask fermentation and 4377 mg/L using a 50 L bioreactor by the engineered FA-11. In conclusion, <i>COMT</i> enzyme engineering combined with global metabolic engineering effectively improved the production of ferulic acid and successfully obtained a fairly high level of ferulic acid production.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 4","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70018","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778276","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":"Meet Our Editorial Board–Engineering in Life Sciences. An Interview With Sascha Beutel Leibniz University Hannover, Hannover, Germany","authors":"Paul Trevorrow,&nbsp;Sascha Beutel","doi":"10.1002/elsc.70019","DOIUrl":"10.1002/elsc.70019","url":null,"abstract":"&lt;p&gt;I am a diploma chemist by profession, working at the Institute of Technical Chemistry. Despite the name, our work primarily focuses on biotechnology, specifically in bioprocessing. This includes both upstream processing of recommended organisms and downstream processing of products like proteins.&lt;/p&gt;&lt;p&gt;We often produce recombinant enzymes, such as those used for the production of flavors or fragrances like terpenes and flavonoids. This involves recombinantly expressing the necessary proteins or enzymes, isolating them, and applying them in various enzyme technical processes. Our area of expertise encompasses both upstream and downstream processes for prokaryotes, as well as sensor development, including optical sensors, fluorescent sensors, and scattered light sensors.&lt;/p&gt;&lt;p&gt;Our institute has a long-term collaboration with industry partners. For example, we have developed the SFR vario together with the company PreSens Precision Sensing GmbH, a tablar for online measurements in shake flasks. Additionally, I have been involved in laboratory digitalization projects aimed at creating more intelligent laboratory infrastructures. These efforts have garnered significant attention, particularly through our involvement in the Labvolution, a major biotechnology trade fair in Hanover, previously known as Biotechnica.&lt;/p&gt;&lt;p&gt;Following the retirement of my former supervisor, Thomas Scheper, I have assumed responsibility for additional projects, including mammalian cell culture for monoclonal antibody production and a collaborative project with the United Kingdom focused on the differentiation and large-scale production of T cells. These projects, while not typically within my usual scope, required continuation and successful completion. Consequently, I have taken on these responsibilities to ensure their advancement.&lt;/p&gt;&lt;p&gt;It is important to be at the right place at the right time, particularly in public services or academia. Personally, I had the opportunity to make this decision while I was a PhD student and already a father of two children. My supervisor at the time, Thomas Sheper, offered me a postdoctoral position upon the completion of my thesis. Considering my family responsibilities, I decided that remaining in public service would be beneficial.&lt;/p&gt;&lt;p&gt;Initially, we agreed that I would take on a steady position without the intention to habilitate. This arrangement lasted for approximately 10–12 years. Eventually, my supervisor prompted me to consider habilitation, which I pursued while maintaining my secure position. This unusual but advantageous situation allowed me to build my research group effectively and complete my habilitation without facing stringent time constraints or deadlines.&lt;/p&gt;&lt;p&gt;The primary reason for choosing public service was to balance professional commitments with family life, making it easier to witness my children's growth compared to working in the private sector.&lt;/p&gt;&lt;p&gt;I enjoy reading and watching movies. I also stay very c","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 3","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11924274/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143669328","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":"Overproduction and Characterization of Recombinant Soluble Trypanosoma brucei Phospholipase A2","authors":"Oluwafemi Abiodun Adepoju,&nbsp;Daniel Quinnell,&nbsp;Harshverdhan Sirohi,&nbsp;Emmanuel Amlabu,&nbsp;Abdullahi Balarabe Sallau,&nbsp;Abdulrazak Ibrahim,&nbsp;Sunday Ene-Ojo Atawodi,&nbsp;Mohammed Nasiru Shuaibu,&nbsp;Geoffrey Chang,&nbsp;Emmanuel Oluwadare Balogun","doi":"10.1002/elsc.70005","DOIUrl":"https://doi.org/10.1002/elsc.70005","url":null,"abstract":"<p><i>Trypanosoma brucei</i> phospholipase A₂ (TbPLA₂) is a validated drug target but the difficulty in expressing its soluble recombinant protein has limited its exploitation for drug and vaccine development for African and American trypanosomiases. We utilized recombinant deoxyribonucleic acid (DNA) technology approaches to express soluble TbPLA₂ in <i>Escherichia coli</i> and <i>Pichia pastoris</i> and biochemically characterize the purified enzyme. Full-length TbPLA₂ was insoluble and deposited as inclusion bodies when expressed in <i>E. coli</i>. However, soluble and active forms were obtained when both the full-length and truncated TbPLA₂ were expressed in fusion with N-terminal FLAG tag and C-terminal eGFP in <i>P. pastoris</i>, and the truncated protein in fusion with N-terminal FLAG tag and C-terminal mClover in <i>E. coli</i>. Truncated TbPLA₂ lacking the signal peptide and transmembrane domain was finally expressed in Rosetta 2 cells and purified to homogeneity. Its migration on sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed its size to be 39 kDa. Kinetic studies revealed that the enzyme has a specific activity of 107.14 µmol/min/mg, a <i>V</i>_max of 25.1 µmol/min, and a <i>K</i>_M of 1.58 mM. This is the first report on the successful expression of soluble and active recombinant TbPLA₂, which will facilitate the discovery of its specific inhibitors for the development of therapeutics for trypanosomiasis.</p>","PeriodicalId":11678,"journal":{"name":"Engineering in Life Sciences","volume":"25 3","pages":""},"PeriodicalIF":3.9,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/elsc.70005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143689016","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}