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:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI-6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI-6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni-NTA agarose. Primary transformed plants harboring the gstI-6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.
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PMID:Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation. 1608 57

Yellow head virus (YHV) is an invertebrate nidovirus that is highly pathogenic for marine shrimp. Nucleotide sequence analysis indicated that the YHV ORF2 gene encodes a basic protein (pI = 9.9) of 146 amino acids with a predicted molecular weight of 16,325.5 Da. The deduced amino acid sequence indicated a predominance of basic (15.1%), acidic (9.6%) and hydrophilic polar (34.3%) residues and a high proportion proline and glycine residues (16.4%). The ORF2 gene was cloned and expressed in Escherichia coli as a M(r) = 21 kDa His(6)-protein that reacted with YHV nucleoprotein (p20) monoclonal antibody. Segments representing the four linear quadrants of the nucleoprotein were also expressed in E. coli as GST-fusion proteins. Immunoblot analysis using YHV polyclonal rabbit antiserum indicated the presence of linear epitopes in all except the V(37)-Q(74) quadrant. Immunoblot analysis of the GST-fusion proteins and C-terminally truncated segments of the nucleoprotein allowed mapping of YHV monoclonal antibodies Y19, Y20 and YII4 to linear epitopes in the acidic domain between amino acids I(116) and E(137). The full-length nucleoprotein was expressed at high level in E. coli and was easily purified in quantity from the soluble cell fraction by Ni(+)-NTA affinity chromatography.
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PMID:Structural and antigenic analysis of the yellow head virus nucleocapsid protein p20. 1621 55

Glutathione transferases (GSTs) are a group of multifunction isoenzymes coded by many genes. A cDNA encoding a novel cytosolic GST enzyme was cloned from a Clonorchis sinensis (Cs) adult worm cDNA library by large-scale sequencing. This new cDNA contains 786 bp with a putative open reading frame of 212 amino acids. The deduced amino acid sequence exhibits 61% identity to C. sinensis cytosolic 28-kDa GST. Recombinant CsGST was overexpressed in Escherichia coli BL21(DE3) and was purified by Ni-NTA Agarose. Enzymatic assays showed that the recombinant CsGST had a high activity of 22.7 U mg(-1). The average K (m) of the CsGST for 1-chloro-2,4-dinitrobenzene is 111 microM. Cibacron blue F3GA and albendazole inhibited the CsGST activity with respective average IC(50) of 1.1 and 247.1 microM. It provides a model for the structure and physiological function analysis on CsGST. The nucleotide sequence reported in this paper has been submitted to the GenBank Database with accession number DQ179264.
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PMID:Molecular cloning and characterization of cDNA encoding a novel cytosolic glutathione transferase from Clonorchis sinensis. 1641 95

Glutathione transferases (GSTs) represent a large family of enzymes. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis (Cs), we isolate another cDNA clone encoding a novel cytosolic GST enzyme. To discriminate with our former reported CsGST, we designated this GST as CsGST1. This new cDNA contains 744 bp with a putative open reading frame of 213 amino acids. The deduced amino acid sequence exhibits 88% identity to Opisthorchis viverrini 28GST (Ov28GST), 60 and 52% identity to C. sinensis cytosolic 28-kDa GST (Cs28GST) and CsGST, respectively. The CsGST1 was expressed in Escherichia coli BL21(DE3) as a His-tag fusion protein and was purified by Ni-NTA agarose. The recombinant CsGST1 showed moderate GST activity of 0.79 U mg(-1). The average K (m) of the CsGST1 for 1-chloro-2, 4-dinitrobenzene is 150 microM. Cibacron blue F3GA and albendazole inhibited the CsGST1 activity with average IC(50) values of 9.1 and 265.4 muM, respectively. The nucleotide sequence reported in this paper was submitted to the GenBank Database with accession number DQ342327.
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PMID:Clonorchis sinensis: molecular cloning and functional expression of a novel cytosolic glutathione transferase. 1704 5

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7-triazacyclononane (tacn), bis(1,4,7-triazacyclononyl) ethane (dtne) and bis(1,4,7-triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu(2+), Ni(2+), Zn(2+), Mn(2+), Co(2+), and Cr(3+), for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C-terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S-transferase-Saccharomyces cerevisiae mitochondrial ATP synthase delta-subunit (GST-deltaATPase-His(6)), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)-nitrilotriacetic acid (im-Ni(2+)-NTA). Investigations using the recombinant GST-deltaATPase-His(6) and recombinant S. japonicum glutathione S-transferase (GST) lacking a hexahistidine tag have confirmed that the C-terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST-deltaATPase-His(6) can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu(2+) or Ni(2+), as IMAC systems.
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PMID:Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from M(N+)-tacn and its derivatives. 1935 Jun 26

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in the Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatography. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2 binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.
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PMID:Purification of caudal-related homeodomain transcription factor and its binding characterization. 2007 18

This study was to acquire recombinant protein of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thromboSpondin type 1 motifs 13), for further studies on its biological function in thrombosis and hemostasis. We transfected the Hela cells with the plasmid pSecTag-ADAMTS13 by lipofectamine. A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. We also measured the enzymatic activity of recombinant protein (rADAMTS13) by GST-His two-site ELISA assay. The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of rADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 kDa with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of rADAMTS13 activity was 6.4 U/mL, according to the normal plasma defined as 1 U/mL. In conclusion, rADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease.
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PMID:[Stable expression and characterization of the von Willebrand factor cleaving protease]. 2043 45

NDK (nucleoside diphosphate kinase) is primarily involved in maintaining cellular nucleotide pools in both prokaryotes and eukaryotes. We cloned ndk from Salmonella typhimurium and expressed it in Escherichia coli as a histidine-tagged protein. The Ni-NTA (Ni(2+)-nitrilotriacetate)-purified protein (sNDK) was found to be tetrameric with a monomeric unit molecular mass of approximately 18 kDa. The sNDK exhibited bivalent-cation-dependent autophosphorylation at a wide range of pH values and the phosphorylation withstands acid or alkali treatment. Surprisingly, nucleoside diphosphates did not behave as 'true inhibitors' of autophosphorylation activity. The sNDK displayed phosphotransfer activity from nucleoside triphosphates to nucleoside diphosphates; however, it was Mg(2+)/Mn(2+)-dependent. Mutational analysis established His(117) as the predominantly phosphorylating residue in sNDK. Although it is a histidine kinase, we found that substitution of Ser(119) with alanine/glutamate significantly affected the autophosphorylation, as well as the NTP-synthesizing ability of sNDK. Interestingly, the mixture of inactive (H117A) and partially active (S119A) proteins was found to be catalytically more efficient than the presence of corresponding amounts of active population, advocating transfer of phosphate from phospho-His(117) to Ser(119). Consistent with this observation, the Ni-NTA-purified H117A protein, obtained following co-expression of both of the mutant constructs [His-tagged H117A and GST (glutathione transferase)-tagged S119A] in E. coli, exhibited autophosphorylation, thereby alluding to intermolecular phosphotransfer between His(117) and Ser(119). Although this housekeeping enzyme has long been discovered and characterized from different sources, the results of the present study portray how Ser(119) in sNDK is phosphorylated. Furthermore, our findings illustrate for the first time that the intermolecular phosphotransfer is mandatory for the efficient NTP synthesis in any NDK.
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PMID:Intermolecular phosphotransfer is crucial for efficient catalytic activity of nucleoside diphosphate kinase. 2057 62

The aim of the study is to obtain an efficient expression of recombinant ubiquitin-like specific protease 1 (Ulp1) by gene engineering. We cloned the Ulp1p, active fragment (403 aa-621 aa) of Ulp1, from Saccharomyces cerevisia, and subcloned into pGEX/Rosetta (DE3) to form an expression plasmid, pGEX-Ulp1p-His6. In order to enhance the solubility of GST-Ulp1p-His6, we purified the fusion protein GST-Ulp1p-His6 by either glutathione S-transferase agarose or Ni-NTA resin chromatography, the purity was up to 98%. We utilized the protein to cleave the SUMO fusions, and the specific activity of GST-Ulp1p-His6 was 1.375 x 10(4) U/mg. This study showed that the recombinant protein GST-Ulp1p-His6 displayed high specificity and activity.
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PMID:[Expression and characterization of soluble recombinant Ulp1p with glutathione S-transferase tag in Escherichia coli]. 2081 66

Inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs), are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. Recent advances in nanotechnology and molecular biology allow the engineering of these peptides with specific affinity to inorganics, often used as molecular linkers or assemblers, to facilitate materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here we report a novel biosynthetic ZnO binding protein and its application in promoting bio-inorganic materials synthesis. In brief, the gene encoding a ZnO binding peptide(ZBP) was genetically fused with His(6)-tag and GST-tag using E.coli expression vector pET-28a (+) and pGEX-4T-3. The recombinant protein GST-His-ZBP was expressed, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Affinity adsorption test demonstrated that the fusion protein had a specific avidity for ZnO nanoparticles (NPs). Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)(2) sol. X-ray diffraction (XRD) analysis on the final products after calcining the precursor powders showed that hexagonal wurtzite ZnO crystals were obtained. Our work suggested a novel approach to the application about the organic-inorganic interactions.
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PMID:Bio-inorganic synthesis of ZnO powders using recombinant His-tagged ZnO binding peptide as a promoter. 2084 4


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