PROTEIN EXPRESSION AND PURIFICATION
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2018
·
IF 1.338
Hualong Xi,Jinfei Yu,Qing Sun,Jingcai Lu,Tiejun Gu,Xiaonan Guo,Bo Li,Xiaorui Chen,Kaixin Zhang,Wei Kong,Yongge Wu
Streptococcus pneumoniae is a major pathogen that causes life-threatening diseases, such as pneumonia, otitis media , bacteremia , and meningitis, worldwide and especially in young children and the elderly. Pneumococcal surface protein A (PspA) is a widely studied candidate protein vaccine that represents a promising replacement for current polysaccharide and polysaccharide-conjugate vaccines. In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography. Using this method, we successfully expressed soluble PspA4 in 10 L of autoinducing culture medium, with a wet-cell yield of 19 g/L and a final PspA4 concentration of 22.8 mg/L. Additionally, we improved PspA4 purity from 17% to 70% in a single step through the use of hydroxylapatite, resulting in acquisition of recombinant PspA4 (>95% purity) at a final yield of 43% from the starting cell-lysis solution. We subsequently verified the secondary structure molecular weight of recombinant PspA4 by circular dichroism and mass spectrometry , respectively. These results demonstrated a highly efficient method for mass producing PspA4 protein and that can also be applied for purification of PspA proteins from other clades.
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ANALYTICAL LETTERS
·
2025
·
IF 1.8
Yu Liao,Yuxin Xue,Haoran Wang,Zhiying Gao,Chao Shi,Cuiping Ma,Yong Li,Yaofang Fan
The application of electrochemical biosensors for detecting foodborne pathogenic bacteria offers benefits like user-friendly, rapid, cost-effective, and portable operation. While screen-printed carbon electrodes (SPCEs) show promise, their rough surfaces and poor stability issues hinder sensitive detection. Here, SPCEs were modified with Fe3O4 and Au nanoparticles to enhance electron transfer. Combined with a chain displacement reaction, a one-pot electrochemical biosensor was developed. It exhibits good reproducibility and stability, detecting Escherichia coli (E. coli) 16S rRNA down to 10−12 M and discriminating single-base mismatches. The biosensor successfully detected E. coli in spiked milk samples, demonstrating its potential for foodborne pathogens detection.
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Synthetic and Systems Biotechnology
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2022
·
IF 4.692
Ming Zhao,Yuting Li,Fengqing Wang,Yuhong Ren,Dongzhi Wei
Itaconic acid (ITA), an effective alternative fossil fuel, derives from the bypass pathway of the tricarboxylic acid (TCA) cycle. Therefore, the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA. The optimization of flux distribution between biomass and production has the potential to the productivity of ITA. Based on the previously constructed strain Escherichia coli MG1655 Δ1-SAS-3 (ITA titer: 1.87 g/L), a CRISPRi-mediated self-inducible system (CiMS), which contained a responsive module based on the ITA biosensor Yp ItcR/P ccl and a regulative CRISPRi-mediated interferential module, was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA. First, a higher ITA-yielding strain, Δ4-P rmd -SAS-3 (ITA titer: 3.20 g/L), derived from Δ1-SAS-3, was constructed by replacing the promoter P J23100 , for the expression of ITA synthesis genes, with P rmd and knocking out the three bypass genes poxB , pflB , and ldhA . Subsequently, the CiMS was used to inhibit the expression of key genes icd , pykA , and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage. The constructed strain Δ4-P rmd -SAS-3 under the dynamic regulation of the CiMS, showed a 23% increase in the ITA titer, which reached 3.93 g/L. This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.
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Foods
·
2026
·Vol. 15
·期 17
·3076
·
IF 6
Hongzhou Chen,Weichao Wu,Mengyu Li,Yang Liu,Chuanfu Lu,Qi Li,Yuwei Ren,Baocai Xu,Yingwang Ye,Kezhou Cai
Foodborne diseases pose a severe threat to human health. As a critical meat-borne pathogen, Escherichia coli O157:H7 ( E. coli O157:H7) can trigger severe illnesses such as hemorrhagic enteritis, threatening public safety and causing a huge economic impact on the meat industry. However, previous detection methods have struggled to strike an optimal balance among accuracy, reliability, and efficiency, with various traditional techniques exhibiting obvious technical shortcomings. To overcome these limitations, this study developed a novel biosensor for the rapid detection of E. coli O157:H7 based on bimetallic carbon-based nanozymes. This sensor leverages the superior peroxidase-like activity and excellent intrinsic fluorescence of bimetallic carbon dots, providing a solid foundation for constructing a dual-modal platform combining colorimetry and fluorescence. Ultimately, this collaborative approach enables the rapid and accurate detection of the pathogen, substantially enhancing the reliability and stability of the results with a limit of detection (LOD) of 10 cfu/mL. This research presents significant practical application value and offers an innovative design strategy for developing advanced diagnostic sensors for E. coli O157:H7 and other meat-borne pathogens.
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Journal of Biological Engineering
·
2026
·Vol. 20
·期 1
·84
·
IF 6.3
Liu Chang,Zhang Hongwei,Xing Wanbin,Na Rina,Meng Xuanyu,Huang Sichen,Wang Linlin,Cao Guoqiang,Wang Pengchao
Background 2-phenylethanol (2-PE) is a high-value aromatic alcohol. Its bioproduction is limited by pathway bottlenecks, limited by a rate-limiting decarboxylation step, product toxicity, and volatilization. We sought to build an efficient Escherichia coli ( E. coli ) platform by optimizing the segment of the Ehrlich route that converts L-phenylalanine (L-Phe) to 2-PE, together with a food-grade soybean-oil overlay for in situ product recovery (ISPR). Results Whole-cell assays showed that phenylpyruvate decarboxylation was the main bottleneck when the yeast decarboxylase Aro10 was used. A phylogeny-guided screen identified Lactococcus lactis KivD as a superior substitute in E. coli . After replacing Aro10 with KivD and tuning expression, leveraging endogenous glutamate dehydrogenase (GDH) for NAD(P)H recycling enabled sufficient cofactor regeneration, yielding 49.5 mM 2-PE from 50 mM L-Phe with 99.0% conversion. At the shake-flask scale, a fed-batch L-Phe feeding strategy coupled with a 2:1 soybean-oil overlay for in situ product removal reduced toxicity and volatilization losses, producing 130 mM 2-PE (15.9 g/L). Using soybean oil as an ISPR phase effectively sequestered 2-PE from the aqueous phase, minimizing volatilization and increasing the overall recovered yield to 94.2% based on L-Phe consumption. Conclusions Replacing the rate-limiting decarboxylation step with KivD, balancing redox through cofactor recycling, and coupling the pathway to a mild oil-overlay recovery increased flux and enabled high-titer 2-PE in E. coli . The workflow of bottleneck substitution plus biocompatible in situ recovery can be transferred to related aromatic alcohols and provides a foundation for de novo routes and high-cell-density fermentations that target still higher titers. Graphical The alternative text for this image may have been generated using AI.
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COMPOSITES PART A-APPLIED SCIENCE AND MANUFACTURING
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2026
·
IF 8.9
Xiaoqian Wu,Junfeng Gu,Weiwei Li,Zelin Liao,Hao Xie,Hao Wang,Weimin Wang,Hang Ping,Zhengyi Fu
Biominerals possess the capability to encapsulate organic macromolecules or inorganic nanoparticles within their matrix crystals during the growth process. The robust interfacial interactions between guest nanoparticles and the host crystals contribute to the exceptional mechanical properties of biominerals. Inspired by this natural encapsulation process, vaterite microspheres incorporating green fluorescence protein-modified Escherichia coli (GFP- E. coli @vaterite) are synthesized. The encapsulation process involves the initial adhesion of amorphous calcium carbonate precursor onto the surface of E. coli , followed by progressive mineralization with growing vaterite crystals, thereby achieving the encapsulation of 14.6 wt% bacteria within vaterite microspheres. Subsequently, dense composite bulks are fabricated through cold sintering of the GFP- E. coli @vaterite powder at 100 °C under a pressure of 1 GPa. The resulting composites achieved flexural strength and fracture toughness of 69.9 MPa and 0.75 MPa·m 1/2 , representing 1.4-fold and 2.2-fold improvements over pure vaterite bulk (48.4 MPa and 0.34 MPa·m 1/2 ), respectively. Notably, the GFP- E. coli @vaterite composite bulks maintained stable fluorescence for more than eighteen months, with minimal signal attenuation and a fluorescence lifetime exceeding 1.1 μs. Our work provides an efficient approach to fabricate integrated structural and functional composites under low temperature conditions. This work also establishes a new direction in fluorescent marking technologies and demonstrates promising potential for practical implementation.
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Micromachines
·
2026
·Vol. 17
·期 2
·151
·
IF 3
Weilin Guo,Yanping Hu,Yunchao Cao,Hongbin Zhang,Hong Wang
Field-effect transistor (FET) biosensors enable label-free and real-time electrical transduction; however, their practical deployment is often constrained by the need for bulky benchtop instrumentation to provide stable biasing, low-noise readout, and data processing. Here, we report a portable extended-gate FET (EG-FET) integrated sensing system that consolidates the sensing interface, analog front-end conditioning, embedded acquisition/control, and user-side visualization into an end-to-end prototype suitable for on-site operation. The system couples a screen-printed Au extended-gate electrode to a MOSFET and employs a low-noise signal-conditioning chain with microcontroller-based digitization and real-time data streaming to a host graphical interface. As a proof-of-concept, enterohemorrhagicEscherichia coliO157:H7 was selected as the target. A bacteria-specific immunosensing interface was constructed on the Au extended gate via covalent immobilization of monoclonal antibodies. Measurements in buffered samples produced concentration-dependent current responses, and a linear calibration was experimentally validated over 104–1010CFU/mL. In specificity evaluation against three common foodborne pathogens (Staphylococcus aureus,Salmonella typhimurium, andListeria monocytogenes), the sensor showed a maximum interference response of only 13% relative to the target signal (ΔI/ΔImax) with statistical significance (p< 0.001). Our work establishes a practical hardware–software architecture that mitigates reliance on benchtop instruments and provides a scalable route toward portable EG-FET sensing for rapid, point-of-need detection of foodborne pathogens and other biomarkers.
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BIOCHEMICAL PHARMACOLOGY
·
2026
·
IF 6.5
Linlin Ding,Rui He,Yongjia Hu,Jian Liu,Paizati Hamidi,Yerzati Nurman,Lei Xu,Jianfeng Wang,Xuming Deng,Aslebek Zekriya,Ying Chen,Qingjie Li
The global spread of multidrug-resistant (MDR) bacteria is devastating health systems and economies worldwide, and this critical trend has revitalized antibiotic adjuvant research. The extensive dissemination of plasmid- or chromosome-mediated resistance genes emphasizes the importance of reshaping colistin sensitivity. Here, we report the discovery that Melissa officinalis essential oil isolated from Traditional Chinese medicine, effectively potentiates the bactericidal activities of colistin against mobile colistin resistance (MCR-1) positive Escherichia coli ( E. coli ). Mechanistic analysis demonstrated that Melissa officinalis essential oil interacts with the MCR-1 protein to disrupt LPS modification, while concurrently inducing microbial iron homeostasis disruption and oxidative damage. To enhance its essential oil-mediated therapeutic potential in clinical management, Melissa officinalis essential oil-loaded nanostructured lipid carrier (MOEO-NLC) was designed with improved biocompatibility and solubility. Encouragingly, the combination of MOEO-NLC and colistin reduced bacterial burden reduction and inflammatory response suppression in a peritonitis-sepsis model. Collectively, this study advances antibacterial tactics and underscores the transformative potential of MOEO-NLC in paving the way for enduring countermeasures against antibiotic-resistant threats.
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LWT-FOOD SCIENCE AND TECHNOLOGY
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2022
·
IF 6.056
Hui-Zhen Zhang,Mei Shu,Wen-Yu Yang,Hong Pan,Men-Xuan Tang,Yuan-Yang Zhao,Chan Zhong,Guo-Ping Wu
The topic of bacteriophage as an alternative to antibiotics to control pathogenic bacteria has attracted attention. Here, a novel virulent Salmonella bacteriophage JNwz02 capable of cross-infecting E. coli O157:H7 was isolated and characterized. The complete genome of JNwz02 was composed of 114390 bp double-stranded DNA molecules encoding 164 open reading frames (ORFs) with a G + C content of 40.22%. JNwz02 belongs to the Siphoviridae family and has high homology with T5-type phages. The eclipse period and latency of JNwz02 infection with Salmonella were 10 min and 10–15 min, respectively. The burst size and adsorption rate constant K were 200 plaque-forming unit (PFU)/colony-forming unit (CFU) and 6.71 × 10 −9 ml/min, respectively. JNwz02 was relatively stable in the range of temperature (30°C-50 °C) and pH (2–11). The viable Salmonella and E. coli O157:H7 were significantly reduced in ready-to-eat duck neck meat and milk after treatment with JNwz02 at 4 °C for 2 d. The results suggested that JNwz02 has potential for application against Salmonella and E. coli in food.
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CHEMOSPHERE
·
2020
·
IF 5.778
Juan Liu,Pingxiao Wu,Fang Wang,Wenchao Niu,Zubair Ahmed,Meiqing Chen,Guining Lu,Zhi Dang
The reuse of polymyxin B (PMB) has attracted extensive attention. Although the resistance mechanism to PMB is clear, there are few reports on the regulation mechanisms and effects of clay minerals on bacteria induced by PMB. The focus of this study is to investigate the multidrug resistance, cell morphology and physiological modification of Escherichia coli ( E. coli ) exposed to PMB in the presence and absence of clay minerals. To be specific, E. coli was cultured serially for 15 days in the increasing concentration of PMB, with or without halloysite or kaolinite . The potential influence mechanisms of halloysite and kaolinite on E. coli was analyzed by proteomics , antibiotic resistance testing, confocal laser scanning microscopy, scanning electron microscopy and Fourier transform infrared. The results showed that kaolinite could obviously promote the growth of bacteria. Moreover, compared with halloysite, kaolinite could stimulate the overexpression of PMB resistance-related proteins ArnA, ArnB and EptA in E. coli exposed to PMB, and promote the synthesis of peptidoglycan and activate glycolysis pathway to produce energy. In contrast, halloysite was able to regulate the production of low molecular weight thiols by E. coli to prevent bacteria from producing excessive reactive oxygen species , activate the oxidative phosphorylation pathway to supply energy for bacterial life activities, and reduce multidrug resistance of E. coli in a variety of ways. These findings are essential for exploring the impacts of clay minerals on the emergence and spread of multi-drug resistant strains in the environment.
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JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY
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2019
·
IF 4.067
Sai Kumar Tammina,Yang Wan,Yanyan Li,Yaling Yang
A simple and one-step microwave digestion method was utilized to synthesize the highly photoluminescent glucosamine derived nitrogen and zinc doped carbon dots (N, Zn-CDs) with a bluish-green luminescence property. The synthesized N, Zn-CDs inherited a good photoluminescence property with a quantum yield of 74% and which were characterized by XPS , XRD , TEM , FT-IR, UV–Vis, and fluorescence spectrometry . More importantly, N, Zn-CDs served as a fluorescence sensor for the detection of Fe 3+ ions, under optimal conditions, a good linear relationship was established in the concentration range of 0.25–125 μM. Also, the experimental results showed the detection limit of N, Zn-CDs against Fe 3+ was 0.15 μM, this could be a promising assay for sensing Fe 3+ ions. Also, the role of zinc doping was explored by conducting the detection under similar experimental conditions in the absence of zinc, and reports revealed that the reason for high photoluminescence was due to the zinc doping. Along with the sensing application, our group had investigated the bactericidal property of synthesized N, Zn-CDs against gram-negative ( Escherichia coli ), and gram-positive pathogens ( Staphylococcus aureus ). N, Zn-CDs displayed good bactericidal activity against both pathogens under light conditions, but Escherichia coli , even under the dark condition proved its activity. Besides, the morphology of both the strains was displayed before and after the treatment via a scanning electron microscopic images. Also, a plausible mechanism for bactericidal activity was also explained. The reported synthesis method could provide a novel approach for the design of high photoluminescent N, Zn–CDs with good sensing and bactericidal property towards Fe 3+ ions, gram-negative and positive bacterial strains .
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APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2014
·Vol. 172
·期 5
·2540-2551
·
IF 1.687
Jia Dongxu,Yang Yu,Peng Zhengcong,Zhang Dongxu,Li Jianghua,Liu Long,Du Guocheng,Chen Jian
Poly(vinyl alcohol) dehydrogenase (PVADH, EC 1.1.99.23) is an enzyme which has potential application in textile industry to degrade the poly(vinyl alcohol) (PVA) in waste water. Previously, a 1,965-bp fragment encoding a PVADH from Sphingopyxis sp. 113P3 was synthesized based on the replacement of the rare codons in Escherichia coli ( E. coli ). In this work, the deduced mature PVADH ( mPVADH ) gene of 1,887 bp was amplified by polymerase chain reaction (PCR) and inserted into the site between Nco I and Hin dIII in pET-32a(+). The constructed recombinant plasmid was transformed into E. coli Rosetta (DE3). In shake flask, the fusion protein of thioredoxin (Trx)-mPVADH was expressed precisely; however, Trx-mPVADH was found to accumulate mainly as inclusion bodies. After isolating, dissolving in buffer containing urea, purification, dialysis renaturation, and digesting with recombinant enterokinase/His (rEK/His), the bioactive mPVADH fragments were obtained with protein concentration of 0.56 g/L and enzymatic activity of 194 U/mL. The K
m and V
max values for PVA 1799 were 2.33 mg/mL and 15.7 nmol/(min·mg protein), respectively. 1 H-NMR and infrared (IR) spectrum demonstrated that its biological function was oxidizing hydroxyl groups of PVA 1799 to form diketone, and PVA 1799 could be degraded completely by successive treatment with mPVADH and oxidized PVA hydrolase (OPH).
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危险品化学品经营许可证（带存储）