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

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.

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.

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.

Development of sandwich-form biosensor to detect Mycobacterium tuberculosis complex in clinical sputum specimens

Mycobacterium tuberculosis, the causing agent of tuberculosis, comes second only after HIV on the list of infectious agents slaughtering many worldwide. Due to the limitations behind the conventional detection methods, it is therefore critical to develop new sensitive sensing systems capable of quick detection of the infectious agent. In the present study, the surface modified cadmium-telluride quantum dots and gold nanoparticles conjunct with two specific oligonucleotides against early secretory antigenic target 6 were used to develop a sandwich-form fluorescence resonance energy transfer-based biosensor to detect M. tuberculosis complex and differentiate M. tuberculosis and M. bovis Bacille Calmette–Guerin simultaneously. The sensitivity and specificity of the newly developed biosensor were 94.2% and 86.6%, respectively, while the sensitivity and specificity of polymerase chain reaction and nested polymerase chain reaction were considerably lower, 74.2%, 73.3% and 82.8%, 80%, respectively. The detection limits of the sandwich-form fluorescence resonance energy transfer-based biosensor were far lower (10 fg) than those of the polymerase chain reaction and nested polymerase chain reaction (100 fg). Although the cost of the developed nanobiosensor was slightly higher than those of the polymerase chain reaction-based techniques, its unique advantages in terms of turnaround time, higher sensitivity and specificity, as well as a 10-fold lower detection limit would clearly recommend this test as a more appropriate and cost-effective tool for large scale operations.

A Progress in Detection of Interactions Between Macromolecules: Linked FRET Using Three Color Fluorophore / 生物化学与生物物理进展

Fluorescence resonance energy transfer (FRET) is the energy transfer from an activated donor fluorophore to a receiving fluorophore. The efficiency of the energy transfer is the function of the distance between the two fluorophores, and is very sensitive to the distance. Its effective response distance is between 1~10 nm, thus it can be used to measure the distance between atoms or molecules. The feature of FRET is very useful in researches on conformational changes, interaction between macromolecules and signal transductions within live cells, and FRET has become a powerful tool in biomedical investigations. However, biological processes often involve interactions between more than two macromolecules, and FRET using two color fluorophores cannot meet the research demand. Recently, two research groups made breakthrough, establishing a novel FRET technique using three color fluorophores based on confocol microscopy and flow cytometry, respectively. The invention will significantly advance researches in biological and related fields.

OPTIMIZATION OF THREE-CUBE FRET ASSAY AND APPLICATION IN THE ANALYSIS OF SUBUNIT ASSEMBLING OF iGluR RECEPTORS IN LIVING CELLS / 解剖学报

Objective To establish and optimize three-cube FRET assay in living cells and analyze subunit assembling of iGluR receptors. Methods Taking HEK293 cells cotransfed with pECFP and pEYFP as negative control, and those transfected with pECFP-YFP as positive control,different calculation methods using fluorescence microscopy were compared. Results These calculation methods were all suitable for FRET measurement in the system. but the measurement results were affected by the ratio of Donor/Acceptor (D/A) in some degree,and different calculation methods have different optimized conditions. FRET measurement using FR value showed subunit specific assembly of iGluR subtypes.Conclusion There are different optimized conditions for these different calculation methods in the three-cube FRET measurement system,and a further evidence is provided for subunit specific assembling of iGluR subtypes from the FRET assay.