Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity / 中国生物制品学杂志

@#Objective To express dengue virus(DENV)NS2B-NS3 protease in E.coli,optimize the expression conditions and determine the enzyme activity,so as to lay a foundation of screening and discovering of lead compounds targeting DENV.Methods Codon-optimized NS2B-NS3 gene was inserted into pET-28a vector to construct recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3,which was transformed E.coli Rosetta(DE3)competent cells and induced by IPTG to express NS2B-NS3 protease. The optimal expression conditions of NS2B-NS3 protease in E.coli were determined by optimizing induction length,induction temperature and IPTG concentration. NS2B-NS3 protease was isolated and purified by HisTrap~(TM) affinity chromatography column and measured for the protease activity by fluorescence resonance energy transfer(FRET)assay.Results The recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3 was constructed correctly as identified by restriction analysis(NheⅠ/XhoⅠ)and sequencing. The optimal expression conditions of NS2BNS3 protease in E.coli were as follows:induction temperature of 20 ℃,induction length of 10 h and IPTG concentration of0. 2 mmol/L. The purified NS2B-NS3 protease showed a purity of more than 90% with a exhibited a of 20 mg/L,which bound to mouse monoclonal antibody against His-tag specifically and had good hydrolytic activity with a specific activity of 16. 111 U/mg,a K_m of 16. 46 μmol/L and a k_(cat) of 0. 028/s.Conclusion DENV NS2B-NS3 protease with high purity and activity was successfully prepared,which laid an experimental foundation of the establishment of high-throughput screening model for inhibitors targeting NS2B-NS3 protease.

Palmitoylation in Regulating Fyn Kinase Activity Based on FRET Imaging / 医用生物力学

Objective To investigate the molecular mechanism of palmitoylation modification in regulating the activity of non-receptor tyrosine kinase Fyn. Methods The intracellular Fyn activity was detected by applying fluorescence resonance energy transfer (FRET) technology, and the mechanism was investigated by combining with Fyn palmitoylation deficiency and C-terminal Src kinase ( CSK ) plasmid co-expression. ResultsExperimental data showed that single loss of either of ( C3, C6) palmitoylation sites resulted in higher Fyn activity, and C6 seemed more significant. It is known that CSK membrane translocation occurred after activation. FRET assay confirmed that CSK could down-regulate the activity of Fyn in cells, but could not effectively regulate the activity of Fyn(GSS) with the loss of palmitoylation sites. Conclusions The results in this study support the hypothesis on Fyn regulation by spatial localization, namely, non-palmitoylated Fyn (GSS) is less effective in the inhibitory regulation by CSK on cell membrane, thus promoting constitutive high activity expression

Progress in research on fluorescence resonance energy transfer technology based on upconversion nanomaterials for biological detection / 中国生物制品学杂志

@#Upconversion nanoparticles(UCNPs)doped with rare earth elements have advantages in biose-nsing because of their good fluorescence stability,biocompatibility and avoidance of background fluorescence. Therefore,fluorescence resonance energy transfer(FRET)system based on upconversion particles(UCNPs based FRET)has been widely used in biological detection. This paper reviews the application and prospect of UCNPs based FRET in biological detection of biotoxins,hormones,proteins,nucleic acids,bacteria,and so on.

Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli / 生物工程学报

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.

Estabishment and evaluation of a new method for rapid detection of CA16 hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer technique / 吉林大学学报(医学版)

Objective: To establish a new method for rapid detection of Coxsachie virus A16 (CA16) hand, foot and mouth disease pathogens based on fluorescence resonance energy transfer (FRET) technique, to evaluate the detection effect and to make the method to meet the requirements of large sample size detection during the outbreak of disease. Methods: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and bicinchoninic acid (BCA) protein assay were used to identify the purity of CA16 chicken yolk antibody (CA16-IgY) and the protein level. Indirect enzyme-linked immunosorbent assay (iELISA) was used to detect the titer and specificity of anti-CA16 IgY antibody. The size, morphology and characterization of gold nanoparticles (AuNPs) and their biological probes (IgY-AuNPs) were determined by UV-visible spectroscopy (UV-Vis), infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The CA16 detection system was constructed based on FRET technique. The sensitivity and specificity of the detection method and clinical sample detection were evaluated by optimizing the IgY-AuNPs concentration, sodium chloride (NaCl) dosage, fluorescence recovery time and other indicators. Results: The CA16-IgY had high purity, the titer was 1:128 000, the average protein level was 12. 15 mg · L-1, and CA16-IgY had good specificity. The results of UV-Vis, FTIR and TEM of AuNPs and IgY-AuNPs showed that IgY was successfully labeled onto the surface of AuNPs, which suggested that IgY-AuNPs could specially recognize CA16 was successfully prepared by electrostatic self-assembly. The CA16 detection system was constructed based on FRET technology, after optimization of the detection system, the optimal dosage of IgY-AuNPs was determined to be 0.52 X 10-3 g · L-1, the optimal dosage of NaCl was 40 μL and the optimal fluorescence recovery time was 90 min. The standard curve of the established detection method was I525 ntu= 15. 452 IgC-9. 746, R2 = 0.993 2, the detection limit was as 1 X 104 PFU · ml-1. Compared with qRT-PCR, the agreement rate reached 93. 75%. Conclusion: A new rapid detection method for CA16 hand, foot and mouth disease pathogens is successfully established, which can be applied to laboratory and clinical tests.

Progress in fluorescent visualization techniques for neurotransmitter detection / 生物工程学报

Neurotransmitters play an important role in nervous system. Temporal and spatial changes of neurotransmitter distribution are crucial to information processing in neural networks. Biosensors that can visually monitor neurotransmitters are one of the vital tools to explore a variety of physiological and pathological activities. This article reviews recent advances in monitoring neurotransmitters with high temporal and spatial resolution, and introduces the latest fluorescent imaging methods for typical neurotransmitters, including glutamate, dopamine, γ-aminobutyric acid and acetylcholine. The article also summarizes the basic principles, advantages and disadvantages of various visually detection methods, and provides systematic suggestions for designing neurotransmitter sensors with high temporal and spatial resolution.

Advances in the research and applications of orange fluorescent protein / 生物工程学报

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

A one-step specific assay for continuous detection of sirtuin 2 activity

Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases with diverse physiological functions. A variety of small molecules have been developed to interrogate the physiological function of SIRTs. Therefore, it is desirable to establish efficient and convenient assays to screen SIRTs modulators. In this study, we designed a series of fluorescent nonapeptide probes derived from substrates of SIRT1-SIRT3. Fluorescence increment of these probes is based on SIRT-mediated removal of the acyl side chain with fluorophore, which makes this system free of lysine-recognizing protease. Comparing the reaction of these fluorescent nonapeptide substrates with SIRT1-SIRT3 and SIRT6, it was confirmed that this assessment system was the most suitable for SIRT2 activity detection. Thus, SIRT2 was used to modify substrates by truncating the amino acids or lysine side chain of nonapeptide. Finally, two specific and efficient fluorescent probes for SIRT2, ne-D9 and ne-K4a, were developed. Evaluation of the results revealed that ne-K4a based assay was more suitable for modulators screening , while the other specific substrate ne-D9 was stable in cell lysate and could detect the activity of SIRT2 in the same. In summary, this study presents a novel strategy for detecting SIRT2 activity and in cell lysate.

Detection of Arginine by AuNPs / CQDs Nanoprobes Based on Fluorescence Resonance Energy Transfer Effect / 分析化学

A novel method for rapid detection of arginine based on fluorescence resonance energy transfer effect (FRET) between carbon quantum dots ( CQDs) and gold nanoparticles ( AuNPs) was developed. Firstly, the CQDs with excellent fluorescence properties were synthesized by one-step microwave assisted method. The AuNPs/ CQDs composites were characterized and their quenching mechanism was analyzed. Then the amount of AuNPs/ CQDs, the pH value and the reaction time were optimal. Under the optimum conditions, the fluorescence system was used to detect the content of arginine, showing a good linear relationship ( R2 = 0. 993 ) between fluorescence intensity and concentration of arginine in the range of 0. 1-10. 0 μmol/ L, and the detection limit was 5. 8 nmol/ L. Finally, the content of arginine in grape juice was determined by this method with recoveries of 105. 4％ -110. 8％ , which indicated that the proposed FRET system had the potential for practical detection of arginine in fruit juice.

Progresses of Single Molecular Fluorescence Resonance Energy Transfer in Studying Biomacromolecule Dynamic Process / 分析化学

Biomacromolecules participate in various kinds of vital processes. Observing and analyzing their structural dynamic and the dynamic processes of intermolecular interaction at molecular level is important for understanding the action mechanism. Since its advent, single molecular fluorescence resonance energy transfer (SM-FRET) has demonstrated its great potential in studying the conformational change and interaction process of biomacromolecules, and a series of new mechanisms have been revealed. This review summarized recent progresses of SM-FRET in studying protein structural dynamic, nucleic acid structural dynamic, protein-protein and protein-nucleic acid interactions.

A Ratiometric Fluorescent Biosensor Based on Exonuclease ⅢAmplification Strategy for Pathogenic Bacterial Gene Detection / 分析化学

Food-borne pathogenic bacteria seriously threaten public health. Based on the mechanism of fluorescence resonance energy transfer (FRET), a ratiometric fluorescence biosensor was constructed by integration of Exo III-based signal amplification strategy. The Cy3 labeled R1-DNA firstly hybridized with Cy5 labeled R2-DNA to form duplex of R1/R2. Cy3 showed a low fluorescence response while Cy5 showed a high fluorescence response. The addition of target pathogenic bacterial gene (Lac Z gene) could de-hybridize the R1/R2,resulting in the fluorescence decreasing of Cy5 and the fluorescence recovering of Cy3. Under the assistance of Exo III, the signal change was further amplified. The detection of limit reached as low as 5.29 pmol/L. The linear detection range was from 10 pmol/L to 2000 pmol/L. The developed ratiomtric detection strategy significantly reduced the possibility of false-positive and false-negative detection results.

Evaluation method for compatibility of compounds in fluorescence resonance energy transfer model to screen β-secretase inhibitors / 中国药理学与毒理学杂志

OBJECTIVE To develop a method to evaluate the compatibility of compounds in the fluorescence resonance energy transfer (FRET) model for β-secretase (BACE1) inhibitor screening.METHODS Two commercially available BACE1 inhibitor screening systems based on FRET were selected to evaluate the BACE1 inhibitory activities of (-)-epigallocatechin-3-gallate (EGCG) and Compound 1 according to the supplier's protocol.The inhibitory rates and slopes of the catalytic curves of the inhibitors were calculated.The effect of inhibitors on the fluorescence intensity of the systems were quantitatively calculated and the comparatively evaluated.RESULTS EGCG,a reported non-competitive inhibitor of BACE1,directly induced the reduction of fluorescence intensity of one of the systems.The slope of the line with the addition of EGCG (10.8±2.6) conformed to that of the line of EGCG inhibition (10.2±3.4),which indicated that EGCG was a pseudo-positive inhibitor of BACE1.Compound 1 had little effect on the fluorescence intensity of the systems,so the inhibitory activity of Compound 1 was confirmed.The compounds which showed inhibitory activity in preliminary screening should be checked in the blank control without BACE1 to calibrate the effect of compound on the system fluorescence intensity.The applicability of the tested compounds in the screening system could thus be evaluated to prevent pseudo-positive results.CONCLUSION This fluorescence calibration method with compound control can be universally used for assays based on FRET theory to evaluate the applicability of tested BACE1 inhibitors.

Insight into the interaction of inhaled corticosteroids with human serum albumin:A spectroscopic-based study / 药物分析学报

It is well known that the safety and efficacy profile of an inhaled cortocosteroid (ICS) is influenced by the pharmacokinetic properties and associated pharmacodynamic effects of the drug. Freely circulating, protein unbound, and active ICS can cause systemic adverse effects. Therefore, a detailed investigation of drug-protein interaction could be of great interest to understand the pharmacokinetic behaviour of corticosteroids and for the design of new analogues with effective pharmacological properties. In the present work, the interaction between some corticosteroids and human serum albumin (HSA) has been studied by spectroscopic approaches. UV–Vis spectroscopy confirmed that all the investigated corticos-teroids can bind to HSA forming a protein-drug complex. The intrinsic fluorescence of HSA was quenched by all the investigated drugs, which was rationalized in terms of a static quenching mechanism. The thermodynamic parameters determined by the Van't Hoff analysis of the binding constants (negativeΔH andΔS values) clearly indicate thathydrogen bonds and van der Waals forces play a major role in the binding process between albumin and betamethasone, flunisolide and prednisolone, while hydrophobic forces may play a major role in stabilizing albumin-triamcinolone complexes.

Liposomal membrane fusion induced by extrusion method / 国际生物医学工程杂志

Objective To study the application of extrusion method in inducing liposome membrane fusion or membrane component mixing,and to investigate the effect of different extrusion conditions on liposome membrane fusion rate.Methods N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) labeled 1,2-dioleoyl lecithin (DOPC) liposomes and non-fluorescently labeled DOPC monolayer liposomes were mixed and extruded.The fluorescence changes before and after the extrusion of the mixed liposomes were observed using laser scanning confocal microscope,and the membrane fusion rate of the mixed liposomes was calculated by fluorescence resonance energy transfer method.Besides,the effects of extrusion times,extrusion pressure and temperature on the fusion rate of liposome membrane were studied.Results The results of laser scanning confocal microscopy showed that the distribution density and intensity of the green fluorescence of N-NBD-PE increased significantly after the extrusion of fluorescently labeled and non-fluorescent labeled DOPC liposomes,which confirmed membrane fusion.After 75 times of extrusion treatments,the liposome membrane fusion rate can reach 26％.The number of extrusions,extrusion pressure and temperature had a significant effect on the fusion rate of the liposome membrane.The higher the number of the extrusions,the smaller the extrusion pressure and the higher the efficiency of the liposome membrane fusion were at physiological temperature.Conclusions Extrusion method can induce liposome membrane fusion and membrane component mixing,and the prepared liposome has a narrower particle size distribution,which is expected to be a new method to induce the bilayer membrane fusion of liposome or lipid vesicle.

High-throughput screening for small molecule inhibitors of the type-I interferon signaling pathway

Interferons (IFNs) are cytokines with fundamental roles in resistance to infections, cancer and other diseases. Type-I IFNs, interferon (IFN-) and interferon (IFN-), act through a shared receptor complex (IFNAR) comprised of IFNAR1 and IFNAR2 subunits. Binding of type-I IFN to IFNAR1 will robustly activate Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Aberrant activation of the type-I IFN response results in a spectrum of disorders called interferonopathies. The purpose of this research is to develop an assay for high-throughput screening (HTS) of small molecule inhibitors of the type-I IFN signaling pathway. Inhibition of type-I IFN signaling can be beneficial in terms of therapeutic use and understanding the underlying mechanism of action. We report here a HTS campaign with the secreted embryonic alkaline phosphatase (SEAP) reporter gene assay against 32,000 compounds which yielded 25 confirmed hits. These compounds were subsequently characterized for their cytotoxicity, effects on STAT phosphorylation and activities in IFN regulatory factor (IRF) transcription.

Research Progresses in Single Molecule Enzymology / 分析化学

The single molecule imaging and technologies that developed in 1990 s have successfully probed the dynamics of single molecule enzyme catalysis in real time in vitro. Ever since then, single molecule enzymology has entered the golden age of rapid developing. Individual features of each enzyme hidden in the overall average have been discovered, and many new catalytic mechanisms have been proposed. Single molecule enzymology sheds light on the dynamic interactions between enzymes and substrates or products, deepening the understanding of biochemical reactions. This review described the recent research progresses of single molecule protease and ribozyme.

Heterodimerization of orexin 2 alpha and 2 beta receptor / 中华行为医学与脑科学杂志

Objective To explore the possibility of heterodimerization between orexin type 2α receptor (OX2αR) and orexin type 2β receptor (OX2βR).Methods Using confocal laser scanning microscope,enzyme linked immunosorbent assay (ELISA),fluorescence resonance energy transfer (FRET) and Bioluminescence resonance energy transfer (BRET) to study the interaction between OX2αR and OX2βR.Result Confocal laser scanning microscope and ELISA showed that OX2αR and OX2βR were both expressed in the cytoplasm.The FRET demonstrated that the signal of the experimental group (OX2αR-YFP+ OX2βR-CFP) was significantly stronger than that of control group (YFP+OX2βR-CFP).The BRET value of the experimental group (OX2αR-YFP+OX2βR-Rluc,mBRET ratio was 65± 15) was higher than that of control group (YFP+ OX2βR-Rluc/OX2αR-YFP+Rluc,mBRET ratio was 10±5) (P＜0.05).Conclusion There are heterodimerization between mOX2αR and mOX2βR.

High salt diet enhances the physical coupling between TRPV4 and cPLA2 / 中国药理学通报

Aim To observe the physical coupling between transient receptor potential channel vanilloid type 4 (TRPV4 ) and cPLA2 in endothelial cells. Methods We investigated the physical association of TRPV4-cPLA2 coupling by immunofluorescence reso-nance energy transfer (immuno-FRET)to assess the spatial proximity between TRPV4 and cPLA2 in human microvascular endothelial cells (HMEC),primary cul-tured endothelial cells and in thoracic aortas rings from high salt-induced hypertension mice.Results At the cellular level,with high salt treatment,the physical in-teraction of TRPV4 and cPLA2 was significantly en-hanced in primary vascular endothelial cells and HMEC.Furthermore, in thoracic aortas rings from high salt-induced hypertension mice,we found an in-creases interaction between TRPV4 and cPLA2 in en-dothelial cells from arterial segments .Conclusion High-salt treatment increases the endothelial TRPV4-cPLA2 coupling,indicating that this coupling may pro-vide a new target for vascular endothelial dysfunction.

Fluorescent Detection of Specific DNA Sequences Related to Toxoplasma gondii Based on Magnetic Fluorescent Nanoparticles Fe3O4/CdTe Biosensor.

Magnetic fluorescent nanoparticles (Fe3O4/CdTe) were prepared in this work and applied for Toxoplasma gondii DNA detection. First, CdTe quantum dots were synthesized with 3- mercaptopropionic (MPA) capped. Fe3O4 magnetic particles were prepared by hydrothermal method with NaOH as precipitator, and they were surfacely modified with silane coupling agent (KH550). After then, the MPA-capped CdTe QDs were immobilized on the Fe3O4 particles surface via electrostatic interaction, and the Fe3O4/CdTe particles were prepared with the average size of 10 nm. The DNA sensing probe was fabricated through labeling a stem-loop Toxoplasma gondii DNA oligonucleotides with Fe3O4/CdTe (donor) at the 5′ end and BHQ2 (acceptor) at 3′ end, respectively. The assembly prosess was verified by UV-Vis, TEM, IR, XRD etc. The sensitivity characterization of the molecular beacon probe was performed by fluorescence spectrum (FS) with a detection limit of 8.339x10-9M. This chemical strategy can be further applied to prepare the magnetic nanoparticles for DNA detection.

Construction and identification of urokinase-type plasminogen activator biosensor plasmid / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet).Methods By the template Src-biosensor, the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5′ end. With the intermediate vector pDMTM-18T, an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and expression of fusion proteins were observed after 24 h.Fluorescence resonance energy transfer (FRET)was observed by the inversion fluorescence microscope and measured by the MetaFlour FRET 4.6 software. Results The uPA biosensor vector was confirmed by the fragment of PCR and double restriction enzyme digestion.The transfection efficiency was nearly 40%.The immunofluorescence detection results displayed that uPA biosensor fusion protein expressed in the 293T cells membrane and the FRET of uPA biosensor in the living 293T cells was observed after incubation with the recombinant human uPA (rhuPA).Conclusion uPA biosensor is successfully constructed and it could be used as a molecular probe to study the temporal and spatial variation of uPA in living cells.