Sheng wu gong cheng xue bao = Chinese journal of biotechnology最新文献

{"title":"[Preparation, optimization, and <i>in vitro</i> evaluation of <i>Pediococcus acidilactici</i> HRQ-1 microcapsules].","authors":"Ruiqin Han, Song Xu, Xinyuan Wang, Jingjing Wang, Xiaoxia Zhang, Liping DU, Zhiyong Huang","doi":"10.13345/j.cjb.240597","DOIUrl":"https://doi.org/10.13345/j.cjb.240597","url":null,"abstract":"<p><p>We have isolated an intestinal probiotic strain, <i>Pediococcus acidilactici</i> HRQ-1. To improve its gastrointestinal fluid tolerance, transportation and storage stability, and slow-release properties, we employed the extrusion method to prepare the microcapsules with <i>P</i>. <i>acidilactici</i> HRQ-1 as the core material and sodium alginate and chitosan as the wall material. The optimal conditions for preparing the microcapsules were determined by single factor and orthogonal tests, and the optimal ratio was determined by taking the embedding rate, survival rate, storage stability, gastrointestinal fluid tolerance, and release rate as the evaluation indexes. The results showed that under the optimal embedding conditions, the embedding rate reached (89.60±0.02)%. Under the optimal formula of freeze-drying protective agent, the freeze-drying survival rate reached (76.42±0.13)%, and the average size of the microcapsules produced was (1.16±0.03) mm. The continuous gastrointestinal fluid simulation experiments confirmed that the microcapsules ensured the viable bacterial count and can slowly release bacteria in the intestinal fluid. The curve of the viable bacterial count during storage at 4 ℃ and room temperature indicated that the prepared microcapsules achieved strains' live number protection. The formula and preparation process of <i>P</i>. <i>acidilactici</i> microcapsules may provide a technological reserve for the preparation of more live bacterial drugs in the future.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1415-1427"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143980727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Serum proteomics and machine learning unveil new diagnostic biomarkers for tuberculosis in adolescents and young adults].","authors":"Yu Chen, Hongxiang Xu, Yao Tian, Qian He, Xiaoyun Zhao, Guobin Zhang, Jianping Xie","doi":"10.13345/j.cjb.240431","DOIUrl":"https://doi.org/10.13345/j.cjb.240431","url":null,"abstract":"<p><p>Adolescents and young adults (AYAs) are one of the major populations susceptible to tuberculosis. However, little is known about the unique characteristics and diagnostic biomarkers of tuberculosis in this population. In this study, 81 AYAs were recruited, and the high-quality serum proteome of the AYAs with tuberculosis was profiled by quantitative proteomics. The data of serum proteomics indicated that the relative abundance of hemoglobin and apolipoprotein was significantly reduced in the patients with active tuberculosis (ATB). The pathway enrichment analysis showed that the downregulated proteins in the ATB group were mainly involved in the antioxidant and cell detoxification pathways, indicating extensive oxidative stress damage. Random forest (RF) and extreme gradient boosting (XGBoost) were employed to evaluate protein importance, which yielded a set of candidate proteins that can distinguish between ATB and non-ATB. The analysis with the support vector machine algorithm (recursive feature elimination) suggested that the combination of apolipoprotein A-I (APOA1), hemoglobin subunit beta (HBB), and hemoglobin subunit alpha-1 (HBA1) had the highest accuracy and sensitivity in diagnosing ATB. Meanwhile, the levels of hemoglobin (HGB) and albumin (ALB) can be used as blood biochemical indicators to evaluate changes in the protein levels of APOA1 and HBB. This study established the serum proteome landscape of AYAs with tuberculosis and identified new biomarkers for the diagnosis of tuberculosis in this population.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1478-1489"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143979025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Research progress in silver ion tolerance mechanisms of <i>Escherichia coli</i>].","authors":"Yuhuang Wu, Xi Zheng, Haoyue An, Shuchu Shen, Zhongbao Wu, Su Zhou, Jun Wang, Lili Zou","doi":"10.13345/j.cjb.240833","DOIUrl":"https://doi.org/10.13345/j.cjb.240833","url":null,"abstract":"<p><p>Due to the wide application of silver-containing dressings and silver-coated medical devices in clinical treatment; the extensive use of antibacterial agents and heavy metal agents in feed factories, <i>Escherichia coli</i> has formed the tolerance to silver ions. To systematically understand the known silver ion resistance mechanisms of <i>E</i>. <i>coli</i>, this article reviews the complex regulatory network and various physiological mechanisms of silver ion tolerance in <i>E</i>. <i>coli</i>, including the regulation of outer membrane porins, energy metabolism modulation, the role of efflux systems, motility regulation, and silver ion reduction. <i>E</i>. <i>coli</i> reduces the influx of silver ions by missing or mutating outer membrane porins such as OmpR, OmpC, and OmpF. It adapts to high concentrations of silver ions by altering the expression of ArcA/B and enhances the efflux capacity of silver ions under high-concentration silver stress via the endogenous Cus system and exogenous Sil system. Furthermore, the motility of bacteria is related to silver tolerance. <i>E</i>. <i>coli</i> has the ability to reduce silver ions, thereby alleviating the oxidative stress induced by silver ions. These findings provide a new perspective for understanding the formation and spread of bacterial tolerance and provide directions for the development of next-generation silver-based antimicrobials and therapies.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1252-1267"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144038786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Construction of novel transmembrane fusion antioxidant enzymes and their protective effect against hydrogen peroxide-mediated cellular oxidative damage].","authors":"Jianru Pan, Ziyi Zhang, Jinnan Chu, Yanan Han, Xueying Zheng, Shirong Cai, Huocong He","doi":"10.13345/j.cjb.240767","DOIUrl":"https://doi.org/10.13345/j.cjb.240767","url":null,"abstract":"<p><p>Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human <i>GST</i> gene to replace the non-human <i>GST</i> gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1547-1558"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144045454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Effects of Gly mutations N-terminal to the integrin-binding sequence on the structure and function of recombinant collagen].","authors":"Fei Li, Yuxi Hou, Ben Rao, Xiaoyan Liu, Yaping Wang, Yimin Qiu","doi":"10.13345/j.cjb.240722","DOIUrl":"https://doi.org/10.13345/j.cjb.240722","url":null,"abstract":"<p><p>Collagen, a vital matrix protein for various tissue and functions in animals, is widely applied in biomaterials. In type Ⅰ collagen, missense mutations of glycine (Gly) in the Gly-Xaa-Yaa triplet of the triple helix are a major cause of osteogenesis imperfecta (OI). Clinical manifestations exhibit marked heterogeneity, spanning a broad disease spectrum from mild skeletal fragility (Type Ⅰ) to severe limb deformities (Type Ⅲ) and perinatal lethal forms (Type Ⅱ). This study utilized recombinant collagen as a model to further elucidate whether Gly→Ala/Val mutations at the N-terminus of the integrin-binding sequence GFPGER affect collagen structure and function, and to explore the underlying mechanisms by which missense mutations impact the biological function of collagen. By introducing Ala and Val substitutions at seven Gly positions N-terminal to the GFPGER sequence, we systematically assessed the effects of these amino acid replacements on the triple-helical structure, thermal stability, integrin-binding ability, and cell adhesion of recombinant collagen. All constructs formed a stable triple-helix structure, with slightly compromised thermal stability. Gly→Val substitutions increased the susceptibility of recombinant collagen to trypsin, which suggested local conformational perturbations in the triple helix. In addition, Gly→Val substitutions significantly reduced the integrin-binding affinity and decreased HT1080 cell adhesion, with the effects stronger than Gly→Ala substitutions. Compared with Gly→Ala substitutions, substitution of Gly with the larger residue Val had enhanced negative effects on the structure and function of recombinant collagen. These findings provide new insights into the molecular mechanisms of osteogenesis imperfecta and offer theoretical references and experimental foundations for the design of collagen sequences and the development of collagen-based biomaterials.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1573-1587"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144043563","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Transcriptomic analysis of suspended Vero cells and reduction of cellular autophagy by epidermal growth factor].","authors":"Muzi Li, Na Sun, Runsheng Peng, Fangfang Ma, Jiamin Wang, Zilin Qiao, Jianguo Chen, Abudureyimu Ayimugl","doi":"10.13345/j.cjb.240530","DOIUrl":"https://doi.org/10.13345/j.cjb.240530","url":null,"abstract":"<p><p>The culture of suspended Vero cells is facing difficulties such as low cell viability and long doubling time. To investigate the main reasons for the slow growth and low viability of suspended Vero cells, this study conducted transcriptomic analysis of suspended Vero cells (Vero-XF) and adherent Vero cells (Vero-AD) to screen the differentially expressed genes (DEGs) affecting the growth of suspended cells. In addition, epidermal growth factor (EGF) was supplemented to the culture system to improve the growth of Vero-XF. The results showed that compared with the Vero-AD group, the Vero-XF group had 7 376 significant DEGs. Kyoto encyclopedia of genes and genomes enrichment analysis revealed that the DEGs were mainly enriched in the autophagy and mitophagy pathways. Eleven DEGs were selected and verified by quantitative real-time PCR, which showed up-regulated expression of <i>ATG9B</i>, <i>WIPI2</i>, <i>LAMP2</i>, <i>OPTN</i>, <i>Rab7a</i>, and <i>DEPTOR</i> and down-regulated expression of <i>ATG4D</i>, being consistent with the results of transcriptomic analysis. In addition, the Vero-XF group showed significantly up-regulated expression of <i>ATG101</i>, <i>ATG2A</i>, and <i>STX17</i> and insignificant change in the expression of <i>NBR1</i>, compared with the Vero-AD group. The protein levels of LC3 and P62 in Vero-XF and Vero-AD were determined by Western blotting, which showed up-regulated expression of LC3Ⅱ/Ⅰ and down-regulated expression of P62 in Vero-XF, indicating a higher level of autophagy. Finally, the exogenous supplementation of EGF at 10, 20, and 30 μg/L in the culture system reduced the autophagy level of Vero-XF by 22.35%, 48.15%, and 71.29%, increased the specific growth rate by 15.48%, 33.33%, and 57.14%, and decreased the apoptosis rate by 2.84%, 15.46%, and 16.23%, respectively. The results of this study preliminarily reveal that the activation of autophagy is one of the reasons for the slow growth of Vero-XF, which provides reference for the subsequent culture of suspended Vero cells.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1671-1689"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144045359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Non-homologous end-joining (NHEJ): physiological function in <i>Mycobacterium</i> and application in gene editing].","authors":"Shasha Xiang, Yu Huang, Jianping Xie","doi":"10.13345/j.cjb.240634","DOIUrl":"https://doi.org/10.13345/j.cjb.240634","url":null,"abstract":"<p><p>DNA double-strand breaks represent a common type of serious DNA damage in living organisms, causing instability of the genome and leading to cell death. Homologous recombination and non-homologous end-joining (NHEJ) are the two main ways to repair DNA double-strand breaks. The core components involved in the NHEJ pathway are highly conserved in both yeast and humans. A few bacteria such as <i>Mycobacterium</i>, <i>Pseudomonas aeruginosa</i>, and <i>Bacillus subtilis</i> also have the NHEJ mechanism. NHEJ plays a key role in the double strand repair of <i>Mycobacterium</i> in latency. This paper summarizes the mechanism and important components of NHEJ in <i>Mycobacterium</i>, introduces the application of NHEJ in gene editing, and reviews the research progress of the NHEJ pathway in <i>Mycobacterium</i>. We hope to bring new insights into the molecular mechanism and provide clues for the application of NHEJ in <i>Mycobacterium</i>.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1280-1290"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Screening and characterization of camelid-derived nanobodies against hemoglobin].","authors":"Ning Zhong, Wenhui Lei, Zuying Liu, Xiaoxiao Xie, Lingjing Zhang, Tengchuan Jin, Minjie Cao, Yulei Chen","doi":"10.13345/j.cjb.240777","DOIUrl":"https://doi.org/10.13345/j.cjb.240777","url":null,"abstract":"<p><p>Hemoglobin, the principal protein in red blood cells, is crucial for oxygen transport in the bloodstream. The quantification of hemoglobin concentration is indispensable in medical diagnostics and health management, which encompass the diagnosis of anemia and the screening of various blood disorders. Immunological methods, based on antigen-antibody interactions, are distinguished by their high sensitivity and accuracy. Consequently, it is necessary to develop hemoglobin-specific antibodies characterized by high specificity and affinity to enhance detection accuracy. In this study, we immunized a Bactrian camel (<i>Camelus bactrianus</i>) with human hemoglobin and subsequently constructed a nanobody library. Utilizing a solid-phase screening method, we selected nanobodies and evaluated the binding activity of the screened nanobodies to hemoglobin. Initially, human hemoglobin was used to immunize a Bactrian camel. Following four immunization sessions, blood was withdrawn from the jugular vein, and a nanobody library with a capacity of 2.85×10⁸ colony forming units (CFU) was generated. Subsequently, ten hemoglobin-specific nanobody sequences were identified through three rounds of adsorption-elution-enrichment assays, and these nanobodies were subjected to eukaryotic expression. Finally, enzyme-linked immunosorbent assay and biolayer interferometry were employed to evaluate the stability, binding activity, and specificity of these nanobodies. The results demonstrated that the nanobodies maintained robust binding activity within the temperature range of 20-40 ℃ and exhibited the highest binding activity at pH 7.0. Furthermore, the nanobodies were capable of tolerating a 10% methanol solution. Notably, among the nanobodies tested, VHH-12 displayed the highest binding activity to hemoglobin, with a half maximal effective concentration (EC₅₀) of 10.63 nmol/L and a equilibrium dissociation constant (K_D) of 2.94×10^-7 mol/L. VHH-12 exhibited no cross-reactivity with a panel of eight proteins, such as ovalbumin and bovine serum albumin, while demonstrating partial cross-reactivity with hemoglobin derived from porcine, goat, rabbit, and bovine sources. In this study, a hemoglobin-specific high-affinity nanobody was successfully isolated, demonstrating potential applications in disease diagnosis and health monitoring.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1515-1534"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143978910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Optimization of promoter screening for heterologous expression of carbonic anhydrase and characterization of its enzymatic properties and carbon sequestration performance].","authors":"Dandan Yao, Yunhui Li, Xingjia Fu, Hui Wang, Yun Liu","doi":"10.13345/j.cjb.240799","DOIUrl":"https://doi.org/10.13345/j.cjb.240799","url":null,"abstract":"<p><p>In this study, high-throughput promoter screening was employed to optimize the heterologous expression of <i>Mesorhizobium loti</i> carbonic anhydrase (MlCA) in order to reduce the costs associated with carbon capture and storage (CCS). To simplify the complexity of traditional vectors, a fusion protein expression system was constructed using superfolder green fluorescent protein (sfGFP) and MlCA. The synthetic promoter library in <i>Escherichia coli</i> was utilized for efficient one-step screening. Based on fluorescence intensity on agar plates, a total of 143 monoclonal colonies were identified, forming a library with varying expression levels. The top four recombinants with the highest fluorescence intensity were selected, among which MlCA driven by the promoter 342042/+ exhibited the highest enzymatic activity, with a specific activity of the 34.6 Wilbur-Anderson units (WAU)/mg. Optimization experiments revealed that MlCA exhibited the best performance when cultured for 4 days under pH 7.0 and 40 ℃ conditions. The Michaelis constant (<i>K</i>_m·hdy) and maximum reaction rate (<i>V</i>_max·hdy) for CO₂ hydration were determined to be 62.46 mmol/L and 0.164 mmol/(s·L), respectively. For esterase hydrolysis, MlCA showed the <i>K</i>_m and <i>V</i>_max of 639.8 mmol/L and 0.035 mmol/(s·L), respectively. MlCA accelerated the CO₂ hydration process, promoting CO₂ mineralized into CaCO₃ within 9 min at low pH and room temperature conditions. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses confirmed that the precipitated product was calcite. This study provides a low-cost and environmentally friendly alternative for future CCS applications.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1588-1604"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144043564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"[Prokaryotic expression of human Alg1 protein and analysis of the transmembrane domain properties].","authors":"Dongzhi Wei, Zhenghui Chen, Chundi Wang, Xiaodong Gao, Ning Wang","doi":"10.13345/j.cjb.240834","DOIUrl":"https://doi.org/10.13345/j.cjb.240834","url":null,"abstract":"<p><p>As the most common type of protein glycosylation, <i>N</i>-glycosylation begins with the synthesis of the dolichol-linked oligosaccharide (DLO) precursor in the endoplasmic reticulum. The mannosyltransferase Alg1 catalyzes the addition of the first mannose molecule to DLO, serving as a key enzyme in this biochemical pathway. The defect of human <i>ALG1</i> gene can lead to the congenital disorders of glycosylation (CDG), i.e., ALG1-CDG. Therefore, it is of great significance to establish the expression and activity assay system of <i>Homo sapiens</i> Alg1 (<i>Hs</i>Alg1) <i>in vitro</i>. In this study, full-length plasmid pET28a-His6-<i>Hs</i>Alg1 and transmembrane domain-lacking plasmid pET28a-His6-<i>Hs</i>Alg1_23-464 were constructed and expressed in <i>Escherichia coli</i>, and the activity of recombinant <i>Hs</i>Alg1 and <i>Hs</i>Alg1_23-464 was measured by liquid chromatography tandem mass spectrometry (LC-MS) with dolichyl-pyrophosphate GlcNAc2 (DPGn2) as the substrate. The results showed that <i>Hs</i>Alg1 had transglycosylation activity, while the activity decreased after protein purification, which was partially restored upon re-addition of membrane components. However, <i>Hs</i>Alg1_23-464 was unable to catalyze glycosylation. The results indicate that the N-terminal transmembrane domain (TMD) of <i>Hs</i>Alg1 plays an important role in the catalytic reaction. This study lays a foundation for further expression and activity analysis of ALG1-CDG-related mutants.</p>","PeriodicalId":21778,"journal":{"name":"Sheng wu gong cheng xue bao = Chinese journal of biotechnology","volume":"41 4","pages":"1535-1546"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144047642","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}