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@#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.
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@#Targeted programmed death-ligand 1 (PD-L1) and CXC chemokine receptor type 4 (CXCR4), gene sequences encoding anti-PD-L1 nanobody and anti-CXCR4 nanobody were cloned into the pET-22b (+) vector to construct recombinant expression plasmid of anti-PD-L1&CXCR4 bispecific nanobody, which was connected with 6 × His tag and transformed into E.coli BL21 (DE3). The expressed proteins were then found to exist as a soluble form in the supernatant of bacterial lysate after induction of IPTG.Three purification methods were used to obtain the target protein in order to improve the yield and purity of the bispecific nanobody.The bacterial supernatant was separated and purified by His Trap FF affinity chromatographic column.The target protein output could exceed 1 mg/L, and the product purity could reach up to 97%.Besides, the anti-PD-L1&CXCR4 bispecific nanobody shows a specific binding ability to two antigens on the cell surface, enhancing the cytotoxicity of IL-2 activated human peripheral blood mononuclear cells (PBMC) to tumor cell line AsPC-1, which lays the foundation for further evaluation of its drug efficacy in vivo.
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【Objective】 To optimize the purification conditions of heparin affinity chromatography in the purification of human coagulation factor Ⅸ by response surface method and establish the optimal chromatography process parameters. 【Methods】 The effect of sample loading temperature on purification efficiency was analyzed through single factor test. Three-factor three-level response surface method was used to optimize the chromatographic elution conditions. The Folin phenol method and the automatic hemagglutination analyzer were used to determine the total protein content and human coagulation factor Ⅸ titer, respectively. The purification effect was evaluated by activity index and process recovery rate. 【Results】 The optimized optimal chromatographic conditions were loading at 5 ℃, washing 4 CV, eluent formulation of sodium citrate 20 mmol/L, arginine hydrochloride 18.7 mmol/L, NaCl 611.6 mmol/L and pH 7.5; under this optimal setting, the recovery rate of the chromatographic process was (46.6±2.9) %, titer of factor Ⅸ rated to (68.4±4.7) IU/mL and specific activity was (62.8±3.3) IU/mg. 【Conclusion】 The optimized parameters of heparin affinity chromatography process by response surface method can produce better purification effect on human coagulation factor Ⅸ.
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Natural products have been a major source of leading compounds in drug discovery. How to effectively screen active compounds from complex matrix remains an interesting topic. In this review, we comprehensively summarized advanced liquid chromatography based approaches in natural products screening, including pre-column, on-column and post-column screening methods. Their advantages, disadvantages and prospect are also discussed.
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OBJECTIVE@#To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide.@*METHODS@#The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B.@*RESULTS@#The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG.@*CONCLUSIONS@#We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.
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Animals , Humans , Rats , Antibodies , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Plasmids , Rats, Sprague-Dawley , Receptor, ErbB-3 , Allergy and Immunology , Recombinant Fusion ProteinsABSTRACT
Affinity chromatography is characterized by its high specificity,high recovery rate and sensitivity,and it has been widely used in the selection of active ingredients of traditional Chinese medicine,separation and enrichment of low molecular weight sugars and protein peptides,research on mechanism of action and discovery of targets.This paper reviewed the application of affinity chromatography and its adsorption isotherm model,kinetic model and adsorption thermodynamic mechanism in the field of traditional Chinese medicine.This summarizes and provides thinking for comprehensive applications of affinity chromatography theory in the field of active ingredient screening,purification and medicine interaction.
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Adsorption , Chromatography, Affinity , Drugs, Chinese Herbal , Chemistry , Medicine, Chinese Traditional , Models, TheoreticalABSTRACT
Objective To construct the prokaryotic expression vector carrying Mycobacterium tuberculosis gene mmpL6 (rv1557), overexpress and purify the recombinant MmpL6 (Rv1557) protein in Escherichia coli (E. coli).Methods The DNA fragment of MmpL6 was amplified by polymerase chain reaction using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template. The target gene was cloned into pET21b vector to construct the pET21b-MmpL6 expression plasmid, and then transformed into E. coli for protein expression. Recombinant protein expression was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and detected by SDS-PAGE combined with Western blot. The overexpressed MmpL6 protein was purified by Ni-affinity chromatography and gel filtration chromatography. Results The recombinant pET21b-MmpL6 plasmid was successfully constructed and the highest expression level was obtained at 25 ℃ in Rosetta strain induced by IPTG. Conclusion The successful expression and purification of MmpL6 in E. coli lays the foundation for the further structure and function studies of the protein, and also provides clues to the design of anti-tuberculosis drugs targeting efflux pump proteins.
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Objective To identify the active components of Andrographis paniculata coupled with Caco-2 cells screened by UPLC-Q-TOF-MS. Methods The cell-combining compounds from A. paniculata were screened based on drug uptake and transport absorption experiment using Caco-2 cells, then the cell dissociation solution was detected before and after treating with A. paniculata by using UPLC-Q-TOF-MS method. The cell-combining compounds from the ethanol extracts of A. paniculata were identified based on the retention time and mass spectrometry information of each chemical composition combined with comparing the extracted ion chromatograms and mass spectrometry datas of reference substances and the related articles. Results A total of 10 compounds combined with Caco-2 cells in ethanol extract of A. paniculata, which were detected and identified as 12,13- dihydroandrographolide (1), skullcapflavone I-2'-O-glucoside (2), andrographolide (3), 14-deoxy-11,12-didehydro andrographiside (4), 7-hydroxy dehydroandrographolide (5), neoandrographolide (6), 3-deoxy dehydroandrographiside (7), deoxyandrographolide (8), dehydroandrographolide (9), and one unidentified component. Conclusion This research establishes a method of screening the active components of A. paniculata coupled with Caco-2 cells using UPLC-Q-TOF-MS analysis, which can be used to screen out the active components of the complex system on traditional Chinese medicines, as well as to lay the foundation for further study of the synergetic compatibility effects among active ingredients.
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Drug-receptor interaction plays an important role in a series of biological effects, such as cell pro-liferation, immune response, tumor metastasis, and drug delivery. Therefore, the research on drug-re-ceptor interaction is growing rapidly. The equilibrium dissociation constant (KD) is the basic parameter to evaluate the binding property of the drug-receptor. Thus, a variety of analytical methods have been established to determine the KD values, including radioligand binding assay, surface plasmon resonance method, fluorescence energy resonance transfer method, affinity chromatography, and isothermal ti-tration calorimetry. With the invention and innovation of new technology and analysis method, there is a deep exploration and comprehension about drug-receptor interaction. This review discusses the differ-ent methods of determining the KD values, and analyzes the applicability and the characteristic of each analytical method. Conclusively, the aim is to provide the guidance for researchers to utilize the most appropriate analytical tool to determine the KD values.
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Objective: To screen and analysis of antihepatic steatosis active components and cell metabolism withinrhubarb anthraquinones in vitro. Methods: Lipid-lowering activity of liver cell affinity components in rhubarbanthraquinones was evaluated by hepatic steatosis in vitro model. Hepatocyte-specific affinity compositions werescreened by hepatocyte affinity chromatography. Hep G2 cells were incubated with rhubarb anthraquinones in vitro andwere detected by HPLC and HPLC-Q-TOF-MS method. Results: Compared with normal group, the intracellular TGcontent of model group were significantly increased (P＜0.05) . Respectively treated with different concentrations ofrhubarb anthraquinones, the intracellular TG content were significantly decreased in a dose dependent manner (P＜0.05) in comparison with model group. Compared with rhubarb solution, there are mainly four hepatocyte-specific affinitycompositions of aloe-emodin, emodin, chrysophanol and physcion, except for rhein. Conclusion: The lipid-lowering experiment in vitro verified that five rhubarb anthraquinones have significant lipid-lowering activity. This research established a biochromatography method to screen out four hepatocyte-specific affinity compositions of aloe-emodin, emodin, chrysophanol and physcion from rhubarb anthraquinones, while the conjugation sites of rhein were ambiguous.Combined hepatocyte affinity chromatography with hepatic steatosis in vitro model, it could evaluate and screen lipidlowering active ingredient in vitro and cell metabolism of Chinese traditional medicine for the drug development of lipidlowering rhubarb anthraquinones.
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Abstract: Background: A key process in cell regulation is protein phosphorylation, which is catalyzed by protein kinases and phosphatases. However, phosphoproteomics studies are difficult because of the complexity of protein phosphorylation and the number of phosphorylation sites. Methods: We describe an efficient approach analyzing phosphopeptides in single, separated protein by two-dimensional gel electrophoresis. In this method, a titanium oxide (TiO2)-packed NuTip is used as a phosphopeptide trap, together with displacers as lactic acid in the loading buffer to increase the efficiency of the interaction between TiO2 and phosphorylated peptides. Results: The results were obtained from the comparison of mass spectra of proteolytic peptides of proteins with a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) instrument. Conclusions: This method has been applied to identifying phosphoproteins involved in the symbiosis Rhizobium etli-Phaseolus vulgaris.
Resumen: Introducción: Un proceso clave en la regulación celular es la fosforilación de proteínas, que se lleva a cabo por cinasas y fosfatasas. Sin embargo, los estudios de fosfoproteómica son difíciles debido a la complejidad de la fosforilación proteica y el número de sitios de fosforilación. Métodos: En el presente trabajo se describe una eficiente estrategia metodológica para analizar fosfopéptidos de proteínas separadas mediante electroforesis bidimensional. En este método, una columna con microesferas de dióxido de titanio (TiO2/NuTip) se utilizó para atrapar los fosfopéptidos en la superficie del TiO2 previamente empacado en una punta. El uso de desplazadores en el buffer de carga, como el ácido láctico, mejoró significativamente la selectividad. Resultados: Los resultados se obtuvieron mediante la comparación de los espectros de masas de péptidos proteolíticos de proteínas analizados utilizando un instrumento de desorción/ionización láser asistida por matriz-tiempo de vuelo (MALDI-TOF). Conclusiones: Este método se ha aplicado para la identificación de fosfoproteínas involucradas en la simbiosis del Rhizobium etli con Phaseolus vulgaris.
Subject(s)
Phosphopeptides/analysis , Phosphoproteins/analysis , Titanium/chemistry , Chromatography, Affinity/methods , Phosphorylation , Rhizobium/metabolism , Symbiosis/physiology , Electrophoresis, Gel, Two-Dimensional/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Phaseolus/metabolismABSTRACT
Objective · To design an immuno-affinity chromatography device for the separation and detection of syphilis specific antibody, then verify its performance of detection and clinical application. Methods · Affinity filler packed by Treponema pallidum (TP) antigen in affinity chromatography can specifically adsorb TP specific antibody (including IgG and IgM) in samples. After balance, elution and desalination, IgG or IgM gold labeled chromatography strip detects the possibly present syphilis specific IgG or IgM antibody. Twenty cases of syphilis antibodynegative samples and 230 cases of syphilis antibody positive clinical specimens were detected by this chromatography device, and 40 cases were also detected by Western blotting.Results ·The standard operation procedure of the affinity chromatography device was optimized, which could effectively detect the specific IgG and IgM antibody of syphilis. The results of 20 syphilis antibody negative samples were all negative. In 230 syphilis antibody positive cases, the detection results were 2 cases with TP-IgG(-) and TP-IgM(-), 210 cases with TP-IgG(+) and TP-IgM(-),10 cases with TP-IgG(-) and TP-IgM(+), and 2 cases with TPIgG(+)and TP-IgM(+). The detection results of 40 cases were compared with the results detected by Western blotting, among which 2 cases detected by affinity chromatography device were TP-IgG(-) and TP-IgM(-), while the results detected by Western blotting were TP-IgG(+) and TP-IgM(-). But the results of the two methods showed no statistically significant difference (P>0.05). Conclusion · The application of the device in separation and detection of IgG and IgM antibodies against TP pathogens is feasible, and it has important value for further application in clinical diagnosis.
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Objective@#To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function.@*Methods@#Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS-PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr-activated SepharoseTM 4B. Reverse high pressure liquid chromatography (HPLC), SDS-PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone-antigen peptide complexes.@*Results@#The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 μg and 1.00 μg HSP70-antigen peptide and 1.00 μg HSP90-antigen peptide activated lymphocytes significantly. Their A490 values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60-antigen peptide and HSP110-antigen peptide did not activate lymphocytes. The killing rates of 1.00 μg HSP70-antigen peptide and 1.00 μg HSP70 were (45.0±2.0)% and (16.0±2.0)%, respectively, showing a significant difference (P=0.012). Similarly, the killing rates of 1.00 μg HSP90-antigen peptide and 1.00 μg HSP90 were (36.0±5.0)% and (13.0±4.0)%, respectively, also showing a significant difference (P=0.048).@*Conclusions@#The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.
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Affinity chromatography (AC)is a type of liquid chromatography that makes use of biological-like interactions for separation and specific analysis of bioactive components. It has been widely used as a high-throughput screening method for the separation,screening and purification of the target molecules from complex samples with advantages such as high selectivity and high recovery efficiency.This article summarizes the biological effects of affinity chromatography, molecular imprinting chromatography, and dye ligands affinity chromatography.The review also encompasses the application of AC in the separation of chiral drugs,screening of active components,purification of target protein,and mechanism of the drug-protein interaction.Moreover,the prospects of its applications are also discussed.
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Affinity chromatography (AC)is a type of liquid chromatography that makes use of biological-like interactions for separation and specific analysis of bioactive components. It has been widely used as a high-throughput screening method for the separation,screening and purification of the target molecules from complex samples with advantages such as high selectivity and high recovery efficiency.This article summarizes the biological effects of affinity chromatography, molecular imprinting chromatography, and dye ligands affinity chromatography.The review also encompasses the application of AC in the separation of chiral drugs,screening of active components,purification of target protein,and mechanism of the drug-protein interaction.Moreover,the prospects of its applications are also discussed.
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This study aims to investigate the targets and targets-involved mechanism for the macrophage activation of low molecular weight saccharides from Cistanche deserticola (LMSC). The phagocytic activity and NO release of RAW264.7 cells were detected, and results showed that LMSC exerts immune activation effect by significantly increasing the phagocytic activity and NO release. LMSC-conjugated epoxy-activated sepharose beads were prepared as affinity reagent to capture the target proteins. Twenty-four proteins such as Eef2 were identified by LC-MS/MS analysis. Pathway enrichment analysis showed LMSC activated RAW264.7 cells by regulating Fcgamma receptor dependent phagocytosis, TNF-alpha NF-κB signaling pathway, glycolysis/gluconeogenesis and the citric acid cycle and respiratory electron transport pathway.
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Drug targets are special molecules that can interact with drugs and exert pharmacological functions in human body. The natural active small molecules are the bioactive basis of traditional Chinese medicine, and the mechanism study is a hot topic now, especially for the identification of their target proteins. However, little progress has been made in this field until now. Here, we summarized the recent technologies and methods for the identification of target proteins of natural bioactive small molecules, and introduced the main research methods, principles and successful cases in this field. We also explored the applicability and discussed the advantages and disadvantages among different methods. We hope this review can be used as a reference for the researchers who engaged in natural pharmaceutical chemistry, pharmacology and chemical biology.
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En este trabajo se implementaron las condiciones para la separación de proteomas de plasma sanguíneo por electroforesis bidimensional. En muestras de plasma de infante y adulto se evaluaron dos sistemas de pretratamiento de la muestra para reducir el rango dinámico de las proteínas: inmunodepleción de proteínas abundantes y enriquecimiento de proteínas de baja abundancia. Los proteomas se separaron por electroforesis bidimensional y las imágenes se analizaron con el programa Melanie 7.0. Se encontró que ambos métodos de pretratamiento fueron reproducibles y permitieron ver las diferencias en los proteomas de infante y adulto, como muestran los análisis de componentes principales y de clasificación jerárquica tipo heatmap. El porcentaje de recuperación de proteínas fue mayor con la inmunodepleción en comparación con el enriquecimiento proteico. Estos resultados permitieron concluir que con la inmunodepleción, se tiene mayor control de las proteínas eliminadas y por tanto menor pérdida de información, lo que permite su aplicación en estudios exploratorios para la identificación de potenciales biomarcadores de enfermedad.
The conditions to separate blood plasma proteomes by two-dimensional electrophoresis were implemented. In plasma samples from infant and adult two sample pretreatment systems to reduce the proteins dynamic range were evaluated: Immunodepletion of abundant proteins and protein-enrichment of low abundance proteins. Proteomes were separated by two-dimensional electrophoresis and the images were analyzed using Melanie 7.0. It was found that both pretreatment methods were reproducible and allowed to see the differences in the proteomes of infant and adult, as evidenced by the principal component analysis and heatmap, a type of hierarchic classification. The percent recovery of proteins was greater with the immunodepletion method, compared to the protein enrichment system. With these results, we conclude that reducing the complexity of plasma by immunoaffinity showed better control of unrecovered proteins and therefore less loss of information, which allows its application on exploratory studies to identify potential biomarkers of disease.
O objetivo deste trabalho foi otimizar as condições para a separação de proteomas do plasma sanguíneo por eletroforese bidimensional para sua aplicação na procura de potenciais biomarcadores. Trabalhou-se com amostras de plasma de crianças e adultos, avaliando dois sistemas de pre-tratamento da amostra para diminuir o espectro dinâmico das proteínas, imunodepleção de proteínas abundantes e enriquecimento de proteínas de baixa abundância. Os proteomas foram separados por eletroforese bidimensional e as imagens foram analisadas com o programa Melanie 7.0. Foi encontrado que ambos métodos de pre-tratamento foram reprodutíveis e permitiram observar as diferenças nos proteomas de crianças e adultos, como foram evidenciadas com as análises de componentes principais e de classificação hierárquica tipo heatmap. A porcentagem de recuperação de proteínas foi maior na imunodepleção do que obtido pelo enriquecimento proteico. Estes resultados permitiram concluir que com a imunodepleção há um controle mais eficiente das proteínas eliminadas e assim uma menor perda de informação, isto permite sua aplicação em estudos exploratórios orientados na identificação de potenciais biomarcadores da doença.
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Background & objectives: Several host defense proteins known to possess antimicrobial activities are present on mucosal surfaces and are consequently found in body fluids of vertebrates. Naturally occurring protease inhibitors like cystatins, especially cystatin C (cys C), are abundantly present in human seminal plasma. Although its antiviral activity against herpes simplex virus (HSV) has been demonstrated, the role of this protein against HIV is not well studied. Therefore, the aim of the present study was to evaluate the anti-HIV activities of cys C, which is present innately in the male reproductive tract. Methods: Protein-protein interaction of cys C with various HIV proteins was studied using a commercially available HIV blot and specific interaction with HIV protease was studied by dot-blot technique using commercially available cys C. to purify biologically active cys C from human seminal plasma to be used for subsequent experiments, gel-permeation chromatography followed by affinity chromatography was used. The HIV infectivity inhibition activity of the purified cystatin C was tested in TZM-bl cells. To study its activity on HIV protease, time-course enzyme kinetics studies were performed using spectrometric assay. Results: Cystatin C reacted with some HIV proteins including HIV protease. Biologically active cys C was purified using gel permeation chromatography followed by affinity chromatography. When tested in TZM-bl cells, purified cystatin C demonstrated HIV-infectivity inhibitory activity (IC50: 0.28 μM). enzyme kinetic studies demonstrated that it abrogated the action of HIV protease on its substrate. Interpretation & conclusions: The present data demonstrate that cystatin C possesses anti-HIV activities. molecular models need to be designed with this protein which would assist towards prevention/ therapeutics against HIV.
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Biological activity evaluation of Chinese materia medica (CMM) composition is one of the main means to explore the active components of CMM. This paper, from the three aspects of whole animal, cellular, and molecular, discusses systematically the technical progress in this area over the past 10 years and the screening of new active ingredients from CMM and monomer compounds based on new technology. At the same time, the different technical strengths and weaknesses have been analyzed in the hope of providing the information and ideas for the research on the new drugs of CMM.