INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Ting Wu,Huashan Sun,Wenyao Wang,Bin Xie,Zhengjie Wang,Jianqi Lu,Anming Xu,Weiliang Dong,Jie Zhou,Min Jiang
Enzymatic PET recycling has emerged as a promising green solution in addition to mechanical recycling, but low soluble expression levels of the inherently hydrophobic PET hydrolases hinder large-scale applications. Here, we propose a novel strategy for enhanced production of FastPETase in Escherichia coli using co-expression of molecular chaperones from Ideonella sakaiensis . Co-expression of cognate DnaK and DnaJ chaperones significantly increased soluble FastPETase expression (up to 2.5-fold), surpassing commercial chaperone plasmids. Furthermore, a combinatorial approach employing co-expression of DnaK/DnaJ chaperones and fusion of FastPETase with the VNp6-tag significantly boosted FastPETase secretion, yielding over 2 g/L of target protein in a 5-l bioreactor. Notably, the crude FastPETase in fermentation broth displayed comparable PET hydrolysis effects to the purified enzyme. This work not only provides new insights into the process of chaperones in protein folding but also suggests a novel and efficient strategy for producing recombinant proteins.
研究领域:
生物化学
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环境科学
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蛋白质工程
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PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2024
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IF 1.4
Dezheng Peng,Yang Li,Linlin Si,Bo Zhu,Peng Wu,Yibang Li,Dongfang Tang,Yu Liu,Yunxiao Zhang
Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys 26 Arg 34 GLP-1 (11–37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity.
研究领域:
药学
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生物技术
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化学工程
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PubMed
高影响因子
MICROCHIMICA ACTA
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2024
·Vol. 192
·期 1
·1-13
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IF 5.3
Liu Junjie,Wu Shengxi,Zhang Lu,Zhang Mingjun,Wu Haotian,Shi Miwan,Weng Yuan,Ye Wenqiao,Gu Kui
A novel proposal is introduced with an unlabeled electrochemical immunosensor for the detection of tumor broad-spectrum biomarker vascular endothelial growth factor (VEGF165) Copper-based metal organic frameworks (Cu MOFs)-carbon nanotubes (MWCNTs) were employed as its substrates, functionalized with methylene blue (MB) for signal enhancement. Cu-MOFs-MWCNTs nanocomposites were synthesized successfully via a solvothermal method and were then deposited on the surface of a glassy carbon electrode (GCE), with the addition of methylene blue to amplify the signal. Due to the expansive specific surface area provided by the carbon nanotubes and the amino groups facilitated by the metal–organic framework nanomaterials, the anti-VEGF165 monoclonal antibody was immobilized on the electrochemical immunosensor through covalent bonding, which could bind specifically to VEGF165, thereby causing a detectable change in the current. The developed immunosensor exhibited a linear detection range for VEGF165 spanning from 1 to 100,000 pg/ml, with the R 2 value of 0.997 by differential pulse voltammetry (DPV) method, and the limit of detection was as low as 0.9686. Additionally, the electrochemical immunosensor presented commendable selectivity, availability, and reproducibility, along with an effective capacity for detecting VEGF165 in clinical samples. Consequently, the immunosensor is expected to revolutionize early screening for VEGF165-related diseases, which offer a promising, cost-effective, and precise diagnostic tool for clinical utility. Graphical
研究领域:
生物医学工程
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肿瘤学
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分析化学
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PubMed
高影响因子
CELL DEATH AND DIFFERENTIATION
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2025
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IF 15.4
Wang Xiaolu,Wang Qianqian,Wang Hong,Cai Guodi,An Yana,Liu Peiqing,Zhou Huihao,Chen Hong-Wu,Ji Shufeng,Ye Jiantao,Wang Junjian
Clinical treatment options for triple-negative breast cancer (TNBC) are currently limited to chemotherapy because of a lack of effective therapeutic targets. Recent evidence suggests that long noncoding RNAs (lncRNAs) encode bioactive peptides or proteins, thereby playing noncanonical yet significant roles in regulating cellular processes. However, the potential of lncRNA-translated products in cancer progression remains largely unknown. In this study, we identified a previously undocumented small protein encoded by the lncRNA LINC02870 . This protein is localized at the endoplasmic reticulum (ER) and participates in ER stress, thus, we named it the endoplasmic reticulum stress protein (ERSP). ERSP was highly expressed in TNBC tissues, and elevated LINC02870 content was correlated with poor prognosis in TNBC patients. Loss of ERSP inhibited TNBC growth and metastasis both in vitro and in vivo. The pro-oncogenic effects of ERSP could be attributed to its selective activation of the IRE1α/XBP1s branch. ERSP enhances the unfolded protein response (UPR) by interacting with XBP1s, facilitating the nuclear accumulation of XBP1s, thereby promoting the expression of ER stress-related genes. These findings highlight the regulatory role of the lncRNA-encoded protein ERSP in ER stress and suggest that it is a potential therapeutic target for TNBC.
研究领域:
肿瘤学
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分子生物学
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癌症研究
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PubMed
高影响因子
MICROCHIMICA ACTA
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2024
·Vol. 191
·期 7
·1-11
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IF 5.3
Liang Pengda,Lv Bei,Chen Ke,Li Dawei
The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25–750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity. Graphical Abstract
研究领域:
生物化学
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分子生物学
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分析化学
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PubMed
高影响因子
MOLECULES
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2025
·Vol. 30
·期 7
·1558
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IF 4.6
Dingtao Tang,Jiajie Quan,Zhengjiao Gao,Bingfeng He,Yu Hou,Peipei Fan,Meidong Pan,Jiali Yang
Prenylation increases the structural diversity and biological activity of flavonoids. In this study, an aromatic prenyltransferase, FgPT1, was identified fromFusarium globosum. This enzyme was demonstrated to specifically catalyze the prenylation of flavanones, including naringenin, hesperitin, eriodictyol, liquiritigenin, rac-pinocembrin, and dihydrogenistein, and exhibited no activity toward other types of flavonoids, including chalcones, flavonols, isoflavonoids, and flavonols. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and nuclear magnetic resonance (NMR) analysis indicated that the majority of prenylated products were 6-Cprenyl flavanones, with the exception of liquiritigenin, which was additionally transformed to 4′-Oprenyl liquiritigenin. Enzyme kinetic analysis suggested that FgPT1 exhibited the highest catalytic efficiency towards naringenin, with akcat/KMvalue determined as 61.92 s−1M−1, and the lowest catalytic efficiency towards liquiritigenin, with akcat/KMof 1.18 s−1M−1. Biochemical characterization suggested that FgPT1 functioned as a metal-dependent enzyme with optimal activity in the presence of Ba2+at pH 7.5 and 30 °C. Site-directed mutagenesis resulted in a series of mutants, including A325V with impaired prenylation activity and V116I, V181I, and V194I with enhanced activity. V194I displayed the highest enzymatic activity with a nine-fold increase compared to wild-type FgPT1.
研究领域:
生物化学
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分子生物学
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PubMed
高影响因子
Current Research in Microbial Sciences
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2024
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IF 4.8
Sirui Zhang,Jingdan Wang,Rong Yu,Haiping Liu,Shuyan Liu,Kai Luo,Jin'e Lei,Bei Han,Yanjiong Chen,Shaoshan Han,E Yang,Meng Xun,Lei Han
Although various mechanisms of carbapenem-resistance have been identified in the nosocomial pathogen Acinetobacter baumannii , the critical process of resistance evolution and the factors involved in are not well understood. Herein, we identified a universal stress protein Usp1413 which played an important role in adaptive resistance of A. baumannii to meropenem (MEM). Based on RNA-Seq and genome sequencing, Usp1413 was not only one of the most downregulated USPs, but also the bare one having mutation of tyrosine and glycine inserted at the site of 229-230 (YG229-230) under the stimulation of MEM. Deletion of Usp1413 resulted in increased MEM resistance. In addition, Usp1413 affected the bacterial abilities of biofilm formation and swarm motility, as well as helped A. baumannii response to various environmental stresses. These effects of Usp1413 were achieved by regulating its interaction proteins, within the functions of YigZ family protein, acetyltransferase, and SulP family inorganic anion transporter. The insertion mutation of YG229-230 influenced both the expression of interaction proteins and the phenotypes of bacteria. Finally, the promotor region of Usp1413 was convinced by point mutations. Overall, our findings identified the universal stress protein Usp1413 as a contributor involved in MEM adaptive resistance and responded to numerous environmental stresses. This study provides novel insights into the mechanism of universal stress proteins in participating antibiotic resistance, and affords a potential target for controlling drug resistance development in A. baumannii .
研究领域:
微生物学
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抗药性研究
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分子生物学
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PubMed
高影响因子
Molecular Catalysis
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2024
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IF 3.9
Yuqiu Wu,Fanjin Meng,Jiaolong Fu,Yuyang Zhou,Huan Fan,Xin Ju,Liangzhi Li
D-xylose, a biomass derivative, is used as a substrate to generate D-xylulose and D-ribulose with high-added value through a cascade reaction. During this process, the thermal stability and catalytic efficiency of rate-limiting enzymes during cascade reactions are important issues that must be addressed in industrial applications. In this study, we prepared and validated PDA/SiO 2 microspheres as highly loaded immobilization carriers by oxidative self-polymerization of polydopamine (PDA) on silica (SiO 2 ) microspheres. D-xylose was successfully catalyzed by the co-immobilized D-5-phosphate ribose isomerase B (RpiB) and D-tagatose-3-epimerase (DTE), denoted PDA/SiO 2 -RpiB-DTE, to produce D-ribulose and D-xylulose. After immobilization, the K m of co-immobilized RpiB and DTE decreased, which suggested increased substrate affinity. The half-life of the co-immobilized enzymes was 3.5–8 fold higher than that of the free enzymes at 50–70 °C, and the inactivation rate constant was reduced, demonstrating that immobilization improved significantly the thermal stability. The conversion of D-xylose at 10 mM was 29.98% under optimal conditions, which contained 9.85% D-ribulose and 20.13% D-xylulose. PDA/SiO 2 -RpiB-DTE showed up to 89.10% initial enzyme activity after 3 recycle runs. The thermal stability and recovery aspects were improved by immobilization, which facilitates the use of biomass to produce high-value-added products and further exploitation.
研究领域:
生物技术
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酶工程
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生化工程
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高影响因子
Nature Communications
·
2025
·Vol. 16
·期 1
·1-19
·
IF 15.7
Qi Houbao,Gao Haiting,Li Meihui,Sun Tianyong,Gu Xiufeng,Wei Li,Zhi Mengfan,Li Zixuan,Fu Dachuan,Liu Yiran,Wei Ziyi,Dou Yu,Feng Qiang
Parvimonas micra ( P. micra ), an opportunistic oral pathogen associated with multiple cancers, has limited research on its role in oral squamous cell carcinoma (OSCC). This study shows that P. micra is enriched in OSCC tissues and positively correlated with tumor metastasis and stages. P. micra infection promotes OSCC metastasis by inducing hypoxia/HIF-1α, glycolysis, and autophagy. Mechanistically, P. micra surface protein TmpC binds to CKAP4, a receptor overexpressed in OSCC, facilitating bacterial attachment and invasion. This interaction activates HIF-1α and autophagy via CKAP4-RanBP2 and CKAP4-NBR1 pathways, driving metastasis. Targeting CKAP4 with masitinib or antibodies impairs P. micra attachment and abolishes P. micra -promoted OSCC metastasis in vitro and in vivo. Together, our findings identify P. micra as a pathogen that promotes OSCC metastasis and highlight that TmpC-CKAP4 interaction could be a potential therapeutic target for OSCC.
研究领域:
肿瘤学
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微生物学
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分子生物学
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癌症研究
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
·
IF 7.7
Bingjie Xu,Dan Yu,Chenlu Xu,Yujie Gao,Hengqiu Sun,Lei Liu,Yang Yang,Dongming Qi,Jindan Wu
Innovative antibacterial therapies using nanomaterials, such as photothermal (PTT) and photodynamic (PDT) treatments, have been developed for treating wound infections. However, creating secure wound dressings with these therapies faces challenges. The primary focus of this study is to prepare an antibacterial nanofiber dressing that effectively incorporates stable loads of functional nanoparticles and demonstrates an efficient synergistic effect between PTT and PDT. Herein, a composite nanofiber mat was fabricated, integrating spherical molybdenum disulfide (MoS 2 ) nanoparticles. MoS 2 was deposited onto polylactic acid (PLA) nanofiber mats using vacuum filtration, which was further stabilized by sodium carboxymethyl cellulose (CMC) adhesion and glutaraldehyde (GA) cross-linking. The composite nanofibers demonstrated synergistic antibacterial effects under NIR light irradiation, and the underlying mechanism was explored. They induce bacterial membrane permeability, protein leakage, and intracellular reactive oxygen species (ROS) elevation, ultimately leading to >95 % antibacterial activity against Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ), which is higher than that of single thermotherapy (almost no antibacterial activity) or ROS therapy (about 80 %). In addition, the composite nanofiber mats exhibited promotion effects on infected wound healing in vivo. This study demonstrates the great prospects of composite nanofiber dressings in clinical treatment of bacterial-infected wounds.
研究领域:
生物医学工程
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纳米医学
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感染与免疫
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DOI
PubMed
高影响因子
ACS Synthetic Biology
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2024
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IF 3.7
Jialing Wang,Jie Chen,Kemin Lv,Zhen Gao,Jiahuang Li,Bin Wu,Bingfang He,Gerhard Schenk
Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2025
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IF 3.9
Yuwen Wei,Fulong Li,Yukun Zheng,Youxiang Liang,Yan Du,Huimin Yu
Transaminases have important applications in the synthesis of drug intermediates such as chiral amines. However, natural transaminases exhibit suboptimal thermal stability, limiting their further applications. Building upon an Rhodobacter sp. -derived ( R )-selective transaminase ( Rb TA), we report a dual-region coupling engineering approach to improve thermostability of Rb TA by strengthening the core hydrogen-bond networks and rigidifying the flexible surface loop. Through single strategy, we identified 4 thermostability improved single mutations, among which I249Q demonstrated the most substantial improvement, achieving a 18-fold increase in half-life ( t 1 / 2 40 ) and a 11.2 ℃ increase in T 50 10 . Then in strategic coupling, the synergistic effect of dual-region modification was observed in both thermal stability and activity enhancement, as mutant with the best high-temperature catalytic performance, R136P/F228Y, had its T 50 10 improved by 7.1℃ and exhibited a 4.2-fold increase in k cat / K m towards ( R )-3-amino-1-butanol. Finally, R136P/F228Y achieved a 20.5 % improvement in conversion over WT in an analytical-scale synthesis in 72 h at a 5 ℃ elevated catalytic temperature. Molecular dynamics simulations demonstrated that the synergy of the formation of new hydrogen bonds and decrease in flexibility accounted for the thermostability improvements. This study provides guidance for enhancing thermostability of similar fold-type enzymes without impairing enzymatic activity in an efficient manner.
研究领域:
生物化学
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蛋白质工程
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）