RESUMO
Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.
Assuntos
Peroxidase do Rábano Silvestre/genética , Antígenos , Clonagem Molecular , Escherichia coli/genética , Peroxidase do Rábano Silvestre/biossíntese , Peroxidase do Rábano Silvestre/metabolismo , Fragmentos Fab das Imunoglobulinas , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object - tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2-4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1-0.2 ml tested solution (extract from 10-20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.
Assuntos
Cromatografia DEAE-Celulose/métodos , Doenças das Plantas/virologia , Vírus do Mosaico do Tabaco/isolamento & purificação , Anticorpos Antivirais/análise , Ensaio de Imunoadsorção Enzimática , Nanopartículas Metálicas , Vírus do Mosaico do Tabaco/imunologiaRESUMO
We present a novel computational ligand-based virtual screening approach with scaffold hopping capabilities for the identification of novel inhibitors of ß-lactamases which confer bacterial resistance to ß-lactam antibiotics. The structures of known ß-lactamase inhibitors were used as query ligands, and a virtual in silico screening a database of 8 million drug-like compounds was performed in order to select the ligands with similar shape and charge distribution. A set of numerical descriptors was used such as chirality, eigen spectrum of matrices of interatomic distances and connectivity together with higher order moment invariants that showed their efficiency in the field of pattern recognition but have not yet been employed in drug discovery. The developed scaffold-hopping approach was applied for the discovery of analogues of four allosteric inhibitors of serine ß-lactamases. After a virtual in silico screening, the effect of two selected ligands on the activity of TEM type ß-lactamase was studied experimentally. New non-ß-lactam inhibitors were found that showed more effective inhibition of ß-lactamases compared to query ligands.
Assuntos
Antibacterianos/química , Descoberta de Drogas , Inibidores de beta-Lactamases/química , Simulação por Computador , Bases de Dados de Compostos Químicos , Modelos QuímicosRESUMO
Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2,000 members: it includes genetically and functionally different bacterial enzymes capable to destroy the beta-lactam antibiotics. The most common are beta-lactamases of molecular class A with serine in the active center. Among them, TEM-type beta-lactamases are of particular interest from the viewpoint of studying the mechanisms of the evolution of resistance due to their broad polymorphism. To date, more than 200 sequences of TEM-type beta-lactamases have been described and more than 60 structures of different mutant forms have been presented in Protein Data Bank. We have considered the main structural features of the enzymes of this type with particular attention to the analysis of key drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed the BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the corresponding forms of beta-lactamases.
Assuntos
Bactérias/enzimologia , beta-Lactamases/química , beta-Lactamases/genética , Antibacterianos , Bases de Dados de Proteínas , Mutação , Estrutura Terciária de Proteína , Serina , Resistência beta-Lactâmica/genéticaRESUMO
The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-ß-lactam ß-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial ß-lactamase TEM-171, with a Ki of 88 µM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A ß-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A ß-lactamases. We also hypothesise that the presented route for finding non-ß-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.
Assuntos
Compostos de Anilina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Acilação , Compostos de Anilina/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Descoberta de Drogas/métodos , Eletroforese em Gel de Poliacrilamida , Fluorescência , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Inibidores de beta-Lactamases/química , beta-Lactamases/químicaRESUMO
Synthesis of b-lactamases is one of the common mechanisms of bacterial resistance to b-lactam antibiotics including penicillins and cephalosporins. The widespread use of antibiotics results in appearance of numerous extended-spectrum b-lactamase variants or resistance to inhibitors. Mutations of 92 residues of TEM type were found. Several mutations are the key mutations that determine the extension of spectrum of substrates. However, roles of the most associated mutations, located far from active site, remain unknown. We have investigated the role of associated mutations in structure of b-lactamase TEM-72, which contain two key mutation (G238S, E240K) and two associated mutations (Q39K, M182T) by means of simulation of molecular dynamics. The key mutation lead to destabilization of the protein globule, characterized by increased mobility of amino acid residues at high temperature of modelling. Mutation M182T lead to stabilization protein, whereas mutation Q39K is destabilizing mutation. It seems that the last mutation serves for optimization of conformational mobility of b-lactamase and may influence on enzyme activity.
Assuntos
Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Resistência beta-Lactâmica , beta-Lactamases/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínios Proteicos , beta-Lactamases/genéticaRESUMO
Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.
RESUMO
We previously reported cloning of cDNAs which encode two granzyme-like serine proteinases (GLP I and GLP II) from rat duodenum. In this paper we present the cDNA sequence for a novel member of the granzyme-like protein family from rat duodenum, GLP III. The amino acid sequence deduced from the cDNA consists of 248 residues and shows 88.2% identity to GLP I and 50.6% identity to GLP II. Comparison of the amino acid sequence of GLP III with sequences of related proteinases reveals the location of the catalytic amino acid triad and enables the prediction of the substrate specificity. Despite close similarity to GLP I, GLP III is expected to demonstrate different substrate specificity due to a substitution of the Arg residue by Glu at the critical position. Northern blot analysis demonstrates that the GLP III transcript is present only in duodenum.
Assuntos
Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/química , DNA Complementar/genética , Duodeno/enzimologia , Expressão Gênica , Granzimas , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Clones of cDNA encoding two serine proteinases were isolated from a cDNA library prepared from rat duodenum mRNA. The deduced amino acid sequences consisted of 248 residues and possessed a high level of homology to one another and to the sequences of granzymes, cathepsin G, and mast cell proteases I and II. Analysis of the enzymes' primary structures allowed the identification of the catalytic amino acid triad and the prediction of the substrate specificity. Northern blotting experiments showed that while one of these proteinases is expressed only in duodenum, the other enzyme is present in duodenum, lung, and spleen. It is supposed that these proteinases may play an important role in the function of an organism's defence systems.
Assuntos
Duodeno/enzimologia , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Expressão Gênica , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/biossíntese , Distribuição TecidualRESUMO
Overlapping cDNA clones containing mRNA for a putative Lon protease (LonHS) were isolated from cDNA libraries prepared from human brain poly(A)+ RNA. The determined nucleotide sequence contains a 2814-bp open reading frame with two potential initiation codons (positions 62-64 and 338-340). The 5'-terminal 337-nucleotide fragment of LonHS mRNA is highly enriched with G and C nucleotides and could direct synthesis of the LonHS N-terminal domain. More likely this region promotes initiation of protein synthesis from the second AUG codon in a cap-independent manner. The amino acid sequence initiated at the second AUG codon includes 845 residues, over 30% of which are identical to those of eubacterial Lon proteases. Residues of the 'A' and 'B' motifs of NTP-binding pattern and a plausible catalytic serine residue are conserved in LonHS. Northern blot analysis revealed LonHS mRNA in lung, duodenum, liver and heart, but not in thymus cells.
Assuntos
Encéfalo/enzimologia , Proteínas de Escherichia coli , Proteínas de Choque Térmico/genética , Protease La , Serina Endopeptidases/genética , Proteases Dependentes de ATP , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Escherichia coli/enzimologia , Humanos , Dados de Sequência Molecular , Ratos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.
Assuntos
Técnicas Biossensoriais/métodos , Peróxido de Hidrogênio/análise , Adsorção , Sequência de Bases , Técnicas Biossensoriais/instrumentação , Cristalização , Primers do DNA/genética , Enzimas Imobilizadas/química , Ouro , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The potential of the method of scanning electron microscopy (SEM) to visualize the results of individual acts of DNA and oligonucleotides hybridization using gold nanoparticles as label was investigated. Molecule of biotin was introduced into DNA or oligonucleotide, and then it was detected in DNA duplex using a conjugate of streptavidin with gold nanoparticles. Effective imaging of DNA duplexes was possible using a conjugate prepared by covalent binding.. The detection limit of the model oligonucleotide of 19 bases was 20 pg.
Assuntos
DNA/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Varredura/métodos , Hibridização de Ácido Nucleico/métodos , Estreptavidina/química , Biotina/química , Ouro/química , Limite de Detecção , Oligonucleotídeos/química , Silício/químicaRESUMO
We present new approaches to improve the efficiency of DNA by scanning probe microscopy using a highly specific hybridization on affine surfaces and nanostructures of gold as a labels. Scanning probe microscopy allows to register of individual acts of hybridization by the detection of gold labels on the surface affinity followed by automatic calculation of the total.
Assuntos
DNA Bacteriano/química , Microscopia de Varredura por Sonda/métodos , Patologia Molecular/métodos , Ouro/química , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos/químicaRESUMO
Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) withFabfragments of antibodies against atrazine. The resulting genetic construction allows us to switch to any other antibody sequence, via the simple re-cloning of variable parts and an additional reporter enzyme. Conjugates were successfully produced in thePichia pastorismethylotrophic yeast expression system. The target activity of the conjugates (both enzymatic and antigen-binding) has been demonstrated by ELISA method.
RESUMO
Models of three-dimensional structures of Fv domains of three antibodies specific to the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) have been derived from the sequence data by comparative modeling. The same binding site cavities were observed in all cases. The most important residue in antigen binding is tyrosine, which serves as a wall of cavity and putatively forms pi-stacking interaction with aromatic moiety of the ligand. Another cavity wall is formed by hydrophobic residues. At the entrance of cavity a glutamate residue is located in 2 of 3 structures. Docking of 2,4-D and its analogs on the models was performed. On the basis of docking results an experimental cross-reactivity data were qualitatively explained. Using results of the modeling, mutation of glutamate to serine or lysine was suggested to eliminate electrostatic repulsion between antibody and ligand and to improve 2,4-D binding efficiency. Target mutations in the antibody binding site were checked on the model.
Assuntos
Ácido 2,4-Diclorofenoxiacético/imunologia , Anticorpos/química , Praguicidas/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Sítios de Ligação , Reações Cruzadas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase.