Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.
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PMID:Cloning and expression of malarial pyrimidine enzymes. 1557 Dec 77

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.
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PMID:A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles. 1758 89

Human thrombomodulin (hTM) is an endothelial cell-associated protein with potent natural anticoagulant activity by converting thrombin from a procoagulant protease to an anticoagulant. ART-123 is a recombinant soluble hTM (amino acid residues 1-498), and we focused on the physical adsorption of ART-123 onto a polymeric biomaterial surface to develop an antithrombogenic blood-contacting material with preventing the denaturation of hTM and the remaining chemical reagents. The adsorption of hTM onto polysulfone (PSF) films was analyzed quantitatively by quartz crystal microbalance analysis. The adsorption constant and the maximum adsorption amount, calculated by the assumption of a Langmuir-type adsorption, showed that hTM adsorbed with a relatively weak interaction onto the PSF film. The hydrophilic protein lysozyme also showed a Langmuir-type monolayer adsorption, although hydrophobic catalase and fibrinogen showed multilayer adsorption accompanying the denaturation. The physically adsorbed hTM showed high coenzymatic activity for the activation of protein C, as well as anticoagulant activity. Furthermore, the surface wettability of the PSF film was easily controllable by the physical adsorption of hydrophobic and hydrophilic bioactive proteins. The physical adsorption of hTM or bioactive proteins onto polymeric biomaterials will be instrumental for developing an antithrombogenic blood-contacting biomaterial, and for controlling the surface properties of biomaterials.
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PMID:Physical adsorption of human thrombomodulin (ART-123) onto polymeric biomaterials for developing an antithrombogenic blood-contacting material. 1758 6

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.
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PMID:Optimization of aptamer microarray technology for multiple protein targets. 1772 65

In this protocol, a fluorescent aptasensor based on magnetic separation for simultaneous detection thrombin and lysozyme was proposed. Firstly, one of the anti-thrombin aptamer and the anti-lysozyme aptamer were individually immobilized onto magnetic nanoparticles, acting as the protein captor. The other anti-thrombin aptamer was labeled with rhodamine B and the anti-lysozyme aptamer was labeled with fluorescein, employing as the protein report. By applying the sandwich detection strategy, the fluorescence response at 515 nm and 578 nm were respectively corresponding to lysozyme and thrombin with high selectivity and sensitivities. The fluorescence intensity was individually linear with the concentration of thrombin and lysozyme in the range of 0.13-4 nM and 0.56-12.3 nM, and the detection limits were 0.06 nM of thrombin and 0.2 nM of lysozyme, respectively. The preliminary study on simultaneous detection of thrombin and lysozyme in real plasma samples was also performed. It shows that the proposed approach has the good character for simultaneous multiple protein detection.
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PMID:Simultaneously fluorescence detecting thrombin and lysozyme based on magnetic nanoparticle condensation. 1957 12

We describe a sensitive and specific sandwich assay for human alpha-thrombin. The assay takes advantage of sandwich binding of two affinity aptamers for increased specificity, gold nanoparticles for signal amplification, magnetic beads for fast magnetic separation, and inductively coupled plasma mass spectrometry for ultrasensitive detection. Other proteins, such as immunoglobulin G, serum albumin, transferrin, fibrinogen, and lysozyme did not show interference with the assay for human alpha-thrombin. The detection limit of human alpha-thrombin was as low as 0.5 fmol, corresponding to 10 pM thrombin in 50 microL, and the dynamic range covered approximately 3 orders of magnitude.
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PMID:Aptamer-linked assay for thrombin using gold nanoparticle amplification and inductively coupled plasma-mass spectrometry detection. 1967 Aug 69

The detection of disease-related proteins in complex biological media demands sensitive, reliable, and low-cost sensing methods. We present here an assay with aptamer-functionalized silica nanoparticles (NPs) as the sensory platform for thrombin detection in blood serum. As a result of specific protein/aptamer interaction, the presence of thrombin induces the primary aptamer-functionalized NPs to form a sandwich structure with fluorescein-labeled secondary aptamer and results in fluorescent NPs. A conjugated polyelectrolyte is further added to the NP solution to amplify the fluorescent signal. The assay shows high specificity for thrombin detection in serum. In addition, a detection limit of 1.06 nM is obtained, which is significantly improved as compared to that for many fluorescence-based thrombin detection assays. Using lysozyme as a second example, the NP-based detection strategy is shown to be generic and could be readily applicable to the detection of other proteins in practical samples.
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PMID:Conjugated polyelectrolyte-sensitized fluorescent detection of thrombin in blood serum using aptamer-immobilized silica nanoparticles as the platform. 1978 11

Jules Bordet, a pioneering immunologist, lived until the dawn of molecular immunology. He was born in Belgium in 1870, obtained a medical degree in 1892, worked at l'Institut Pasteur in Paris from 1894 to 1901 and then established the Pasteur Institute of Brabant in Brussels. Before World War I, Bordet found that complement binds to antibody-antigen complexes regardless of the antigen or antibodies involved. Subsequently he developed the complement fixation test that was of diagnostic importance for several decades. For his research concerning complement he was awarded the 1919 Nobel Prize in Physiology or Medicine. During that period he also discovered anaphylatoxin, conglutinin, and the cause of whooping cough (Bordetella pertussis). After World War I he found how thrombin forms, how platelets participate in clotting, lysozyme in human milk and much of the biology of bacteriophages. In addition, Bordet worked fervently to limit weapons of mass destruction and promote peace until his death in 1961.
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PMID:Jules Bordet (1870-1961): a bridge between early and modern immunology. 2002 83

Herein, we report two simple label-free electrochemical aptasensors for protein detection using layer-by-layer (LBL) self-assembled multilayers with ferrocene-appended poly(ethyleneimine) (Fc-PEI), carbon nanotubes (CNTs) and aptamer. In one sensing strategy, the Fc-PEI, CNTs and DNA aptamer are LBL assembled on the electrode surface via electrostatic interaction. In the presence of target, the aptamer on the outermost layer of the LBL self-assembled multilayer would catch the target on the electrode interface, which makes a barrier for electrons and inhibits the electro-transfer, resulting in the decreased DPV signals of Fc-PEI. Using this strategy, a wide detection range (0.3-165 ng ml(-1)) for model target thrombin is obtained, with a low detection limit of 0.14 ng ml(-1). In the similar sensing strategy for detection of lysozyme, a wide detection range (0.2 ng ml(-1) to 1.66 microg ml(-1)) and a low detection limit (0.17 ng ml(-1)) are obtained. These results prove that the LBL sensing strategies developed possess sensitivity, selectivity, stability and generality.
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PMID:Layer-by-layer electrochemical biosensor with aptamer-appended active polyelectrolyte multilayer for sensitive protein determination. 2010 54

With the aptamer-induced self-assembly feature of NIR-emissive platinum(ii) terpyridyl complex, a "proof-of-principle" concept in label- and immobilization-free probing strategies of lysozyme and thrombin has been demonstrated with good selectivity and specificity.
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PMID:Aptamer-induced self-assembly of a NIR-emissive platinum(II) terpyridyl complex for label- and immobilization-free detection of lysozyme and thrombin. 2087 76


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