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)

To investigate the failure of high-level production of hepatitis B viral (HBV) surface antigen (HBsAg), including three authentic forms, large (L), middle (M) and major/small (S) HBsAg, in Escherichia coli, we employed the high-expression vector pGEX containing the glutathione S-transferase-encoding gene (GST) to study HBsAg production. Different fragments of HBV DNA containing the entire pre-S1/pre-S2/S region (for L protein), or partial pre-S1, pre-S2, pre-S1/pre-S2 and pre-S2/S region (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PAGE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion proteins, in contrast to those cells harboring plasmids without the S region (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3-10% of the total cellular protein. Using an immunoblot method to screen HBsAg production in cells which harbored plasmids derived from exonuclease BAL 31-digested pGLS, we obtained eight positive clones. Nucleotide sequence analyses of plasmids from the positive clones revealed that termination, deletion or frameshift occurred at the regions encoding either the first or the third transmembrane domain of the major HBsAg. Correlation between the production level of GST-HBsAg fusion proteins and their constituent and arrangement of amino acids (aa) at the last 20 aa among 15 clones suggested that the fusion protein ended with a longer stretch of or a higher ratio of hydrophobic aa had a lower production in E. coli.
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PMID:Deletion or alteration of hydrophobic amino acids at the first and the third transmembrane domains of hepatitis B surface antigen enhances its production in Escherichia coli. 764 92

A family of tyrosine receptor kinases known collectively as trk receptors plays an essential role in signal transduction mediated by nerve growth factor and related neurotrophins. To localize the major trk receptors (trkA, B and C) in the developing and adult central (CNS) and peripheral (PNS) nervous system, we generated monoclonal antibodies (MAbs) to extracellular (MAbs E7, E13, E16, E21, E29) and intracellular (MAb I2) domains of human trkA fused to glutathione S-transferase. Several MAbs (E7, E13, E16) recognized glycosylated trkA (gp140trk and gp110trk) in Western blots, one MAb (E7) recognized non-glycosylated (p80trk) and glycosylated trkA in immunoprecipitation assays, and two MAbs (E13, E29) detected trkA on the cell surface of NIH3T3 cells transfected with a trkA cDNA. Although generated to trkA fusion proteins, this panel of MAbs also recognized trkB and trkC in flow cytometric studies of NIH3T3 cells transfected with trkB or trkC cDNAs. Thus, we used these pan-trk MAbs to probe selected regions of the CNS and PNS including the hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and dorsal root ganglion (DRG) to localize trkA, B, and C receptors in the developing and adult human nervous system. These studies showed that trk receptors are expressed primarily by neurons and are detectable very early in the developing hippocampus, cerebellum, spinal cord, and DRG. Although the distribution and intensity of trk immunoreactivity changed with the progressive maturation of the CNS and PNS, immunoreactive trk receptors were detected in neurons of the adult human hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and DRG. This first study of trk receptor proteins in the developing and adult human CNS and PNS documents the expression of these receptors in subsets of neurons throughout the developing and adult nervous system. Thus, although the expression of trk receptor proteins is developmentally regulated, the constitutive expression of these neurotrophin receptors by neurons in many regions of the adult human CNS and PNS implies that mature trk receptor-bearing neurons retain the ability to respond to neurotrophins long after terminal neuronal differentiation is complete.
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PMID:Expression of trk receptors in the developing and adult human central and peripheral nervous system. 764

The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.
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PMID:The precursor of pea ferredoxin-NADP+ reductase synthesized in Escherichia coli contains bound FAD and is transported into chloroplasts. 765 8

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.
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PMID:Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB. 765 Nov 40

Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of HIV-1 protease expression in different fusion forms. 766 45

The herpes simplex virus 1 UL10 gene encodes a hydrophobic membrane protein dispensable for viral replication in cell culture (J.D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). We report the following. (i) A fusion protein consisting of glutathione S-transferase fused to the C-terminal 93 amino acids of the UL10 protein was used to produce a rabbit polyclonal antiserum. The antiserum reacted with infected-cell proteins which formed in denaturing polyacrylamide gels a sharp band (apparent M(r) of 50,000) and a very broad band (M(r) of 53,000 to 63,000). These bands were not formed by lysates of UL10- virus or by lysates of infected cells boiled in the presence of sodium dodecyl sulfate before electrophoresis. (ii) The proteins forming both bands were labeled by [3H]glucosamine, indicating that they were glycosylated. (iii) The UL10 protein in cells treated with tunicamycin formed a single band (apparent M(r) of 47,000) reactive with the anti-UL10 antibody, indicating that the 47,000-M(r) protein was a precursor of N-glycosylated, more slowly migrating forms of UL10. Treatment of the immunoprecipitate with endoglycosidase H increased the electrophoretic mobility of the 50,000-M(r) species to that of the 47,000-M(r) species, indicating that the 50,000-M(r) species contained high-mannose polysaccharide chains, whereas the proteins forming the 53,000- to 63,000-M(r) bands contained mature chains inasmuch as they were resistant to digestion by the enzyme. (iv) The UL10 protein of R7221 carrying a 20-amino-acid epitope formed only one band with an M(r) of 53,000. This band was sensitive to endoglycosidase H, suggesting that the epitope inserted in the R7221 UL10 protein may have interfered with glycosylation. (v) The UL10 protein does not contain a cleavable signal sequence inasmuch as the first UL10 methionine codon was reflected in the 50,000-M(r) protein. (vi) The UL10 protein is present in virions and plasma membranes of unfixed cells that were reacted with the polyclonal rabbit antibody. In accordance with the current nomenclature, the UL10 protein is designated glycoprotein M.
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PMID:The UL10 gene of herpes simplex virus 1 encodes a novel viral glycoprotein, gM, which is present in the virion and in the plasma membrane of infected cells. 767 47

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.
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PMID:Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. 768 42

The nm23 gene products/nucleoside diphosphate (NDP) kinase expression in prostate carcinomas and benign hyperplasias was evaluated immunohistochemically. Monoclonal antibodies against nm23-H1 and nm23-H2 proteins were prepared using the corresponding proteins fused with glutathione S-transferase as immunogens. Of the 80 cases of nonmetastatic prostate carcinoma examined, 74% (59/80) and 60% (48/80) were immunoreactive for nm23-H1 or nm23-H2 protein, respectively. Negative staining for nm23-H1 occurred in 83% of metastatic lesions, while 34% were negative for nm23-H2. All primary tumors corresponding to the metastases examined showed positive immunostaining for nm23-H1, indicating an inverse relationship between expression of this protein and metastatic status. nm23-H2 protein was detected in 83% of primary tumors and its expression appeared to be significantly correlated to the degree of histological differentiation. In contrast, all cases of benign prostatic hyperplasia showed elevated levels of both nm23-H1 and nm23-H2 expression. These data suggest that the nm23/NDP kinase may play a role in suppressing the expression of malignant potential in prostate carcinomas.
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PMID:Expression of nm23-H1 and nm23-H2 proteins in prostate carcinoma. 769 35

To gain insight into mechanisms of cell type-specific transcription of class mu-glutathione S-transferase genes, the gene encoding the Yb3 subunit was cloned. Yb3 subunits are selectively expressed at high levels in rat brain and testis but not in liver or kidney. The Yb3 subunit gene spans over 6 kb and consists of 8 exons and 7 introns and a sequence consisting of tandem direct repeat consensus octamer DNA binding motifs separated by a 6 base pair (bp) spacer was identified in its 5'-flanking region. Gel shift assays with a 40 bp segment of DNA containing the two consensus octamer sequences, revealed the presence of specific binding proteins in nuclear extracts of rat brain, testis and C6 glioma cells. DNA binding activity was greatly reduced in liver, kidney and HTC cells. Reporter vectors carrying segments of the 5'-flanking region of the Yb3 subunit gene fused to a luciferase gene were introduced into C6 glioma cells which express high levels of Yb3 subunits, and into HTC cells which do not. The plasmids consisting of the Yb3 gene promoter up to, but not including, the octamer motifs did not support luciferase transcription in the C6 glioma cells, but larger fragments that included the octamer repeat sequences, effectively directed transcription in the C6 glioma cells. With mutated octameric sequences transcriptional activity was greatly reduced, and none of the same Yb3 constructs directed substantial luciferase transcription in the HTC cells. The results show that octamer motifs in the 5'-flanking region of the Yb3 subunit gene are functional and are the principal cis-acting elements that account for its discrete cell type-selective expression. This gene is one of the few known targets for octamer DNA binding transcription factors in brain.
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PMID:Brain and testis selective expression of the glutathione S-transferase Yb3 subunit is governed by tandem direct repeat octamer motifs in the 5'-flanking region of its gene. 770 76

The MPS1 gene has been previously identified by a mutant allele that shows defects in spindle pole body (SPB) duplication and cell cycle control. The SPB is the centrosome-equivalent organelle in the yeast Saccharomyces cerevisiae, and it nucleates all the microtubules in the cell. We report the isolation of the MPS1 gene, which encodes an essential protein kinase homolog. The MPS1 open reading frame has been fused to those that encode the LexA protein or the GST protein and both of these constructs function in yeast. The fusion proteins have been affinity-purified from yeast extracts and the GST chimeric protein has been found to be a phosphoprotein. Both proteins have been used to demonstrate intrinsic in vitro protein kinase activity of Mps1p against exogenous substrates and itself (autophosphorylation). A mutation predicted to abolish kinase function not only eliminates in vitro protein kinase activity, but also behaves like a null mutation in vivo, suggesting that kinase activity contributes to the essential function of the protein. Phosphoamino acid analysis of substrates phosphorylated by Mps1p indicates that this kinase can phosphorylate serine, threonine and tyrosine residues, identifying Mps1p as a dual specificity protein kinase.
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PMID:Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase. 773 18


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