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)

Human immunodeficiency virus 1 (HIV-1) nucleocapsid protein p15 was produced as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Rapid purification of GST::p15 in an active form by one-step glutathione-agarose chromatography was accomplished in the presence of an antioxidant. Recombinant p15 fused to GST was shown to stimulate the dimerization of viral RNA. HIV-1 reverse transcriptase-catalyzed in vitro synthesis of minus-strand cDNA from synthetic human tRNA(Lys3UUU) and natural bovine tRNA(Lys3SUU) primer molecules was enhanced by GST::p15. GST produced in E.coli revealed no effect with respect to RNA dimerization and cDNA synthesis, demonstrating that both activities reside in the p15 portion of the fusion protein.
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PMID:Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA. 128 Feb 40

A 167 amino acid fragment of the N-terminal domain of the human type I corticosteroid (mineralocorticoid) receptor was fused to the glutathione S-transferase gene using the Gex expression plasmid and the fusion protein used to raise the monospecific polyclonal antibody, MINREC4. Immunostaining experiments showed that MINREC4 specifically bound type I receptor in the distal tubule of the kidney, the ductal elements of the salivary glands and the epithelium of the distal colon in the rat. Adrenalectomy abolished staining in the parotid and colon, and reduced immunoreactivity in the submandibular gland. Administration of corticosterone or aldosterone resulted in partial restoration of immunostaining in the parotid, and a complete restoration of staining to intact levels in the submandibular gland and colon. These results suggest that adrenocorticoid binding to the type I receptor may result in tissue specific conformational changes in the binding protein and that the MINREC4 antibody may be used to study the effects.
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PMID:Type I corticosteroid receptor-like immunoreactivity in the rat salivary glands and distal colon: modulation by corticosteroids. 132 51

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.
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PMID:Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion. 132 2

The genome of CT10 avian sarcoma virus encodes a 47-kDa fusion protein that consists of viral gag sequences fused to a cell-derived sequence containing SH2 and SH3 domains (v-crk). Genetic and biochemical evidence suggests that v-Crk can induce transformation of chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulation. In this report, we have developed an in vitro microtiter assay to study the binding of bacterially expressed glutathione S-transferase-fusion proteins of v-Crk and its cellular homolog, c-Crk, to the phosphorylated epidermal growth factor receptor (EGFR). Competitive binding data are presented that compare the abilities of heterologous glutathione S-transferase-fusion proteins containing GAPSH2[N], AblSH2, SrcSH2, and PLC-gamma SH2[N] sequences to inhibit Crk binding. Results indicate that both full-length Crk and GAPSH2[N] bind the phosphorylated EGFR with high affinity and can quantitatively compete the binding of each other by competitive enzyme-linked immunosorbent assay. Binding of full-length Crk or the isolated SH2 domains of GAP or Abl resulted in a significant protection of phosphorylated EGFR against dephosphorylation by cellular phosphatase activity, but did not appear to stimulate the intrinsic tyrosine kinase activity of the EGFR. To extend these findings to p130, the major phosphotyrosine-containing protein in CT10-transformed cells, we utilized a nitrocellulose filter binding assay. Results demonstrate high affinity binding of Crk toward denatured p130 and, as is the case for phosphorylated EGFR, Crk binding can partially protect p130 from phosphatase activity. However, no apparent competition of Crk binding was noted with heterologous SH2-containing proteins including GAPSH2[N], suggesting a possible specificity of Crk-p130 binding. These data are consistent with a direct role of SH2 in the modulation of cellular phosphotyrosine status in vivo.
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PMID:Tyrosine-phosphorylated epidermal growth factor receptor and cellular p130 provide high affinity binding substrates to analyze Crk-phosphotyrosine-dependent interactions in vitro. 137 24

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.
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PMID:Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme. 140 Feb 28

Glutathione transferase P (GST-P) gene is specifically and highly activated during rat chemical hepatocarcinogenesis. We have previously cloned the GST-P gene and have identified putative regulatory regions. To further explore regulatory mechanisms, deletion constructs of the GST-P gene fused to the chloramphenicol acetyltransferase (CAT) structural gene were introduced into primary cultured rat hepatocytes by electroporation, and their activity was determined. The expression of the GST-P-CAT fusion gene is quite low in these cells as compared to that in both a rat fibroblast cell line, 3Y1 cells, and a rat hepatoma cell line, dRLh84. The presence of the strong enhancer GPEI did not elicit any enhancing activity at its original position, or when it was located 3' of the CAT gene, although this element does enhance CAT activity significantly when located adjacent to the promoter. Cotransfection of neither c-jun nor c-fos expression vector, nor both vectors, could enhance the CAT activity, even though GPEI consists of two phorbol ester response element-like sites. Furthermore, the expression of jun family gene was not correlated with GST-P gene expression either in primary cultured hepatocytes or in hepatoma cell lines.
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PMID:Analysis of glutathione transferase P gene regulation with liver cells in primary culture. 144 99

For unknown reasons, levels of expression of foreign genes inserted into expression vectors in Escherichia coli have frequently been undetectable. The most critical step in the successful production of foreign proteins seems to be the initiation of translation. Since most prokaryotic genes are transcribed in a polycistronic form, we have devised a new prokaryotic expression system utilizing dicistronic gene organization. Downstream from a strong promoter and the gene encoding glutathione S-transferase from Schistosoma japonicum, various foreign genes were connected via a ribosome-binding site, a stop codon and a start codon. The VH domain of an immunoglobulin fused to the alpha subunit of tryptophan synthase, FK506-binding protein, cyclophilin, and a domain of a major histocompatibility complex antigen were successfully produced in E. coli as discrete polypeptides by this method.
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PMID:Development of a prokaryotic expression vector that exploits dicistronic gene organization. 151 88

The regulation of the Erk (extracellular-signal-regulated kinase) gene-encoded protein kinase activity by reversible phosphorylation has been reported to involve either an activator of autophosphorylation or an upstream protein kinase. In this communication we describe assays utilizing the Erk-1 protein fused to glutathione S-transferase that permit the identification of protein kinase(s) that phosphorylate and activate the myelin basic protein kinase activity encoded by the Erk-1 gene. A phorbol ester-stimulated protein kinase activity was identified that phosphorylated a kinase-negative Erk-1 gene product on tyrosine and threonine. The protein kinase phosphorylated and activated wild-type protein expressed in bacteria from 20- to 50-fold. The activation of the Erk-1-encoded myelin basic protein kinase required ATP and correlated directly with the degree of phosphorylation on the same amino acid residues previously shown to be phosphorylated in vivo. Conversion of the tyrosine site of phosphorylation to phenylalanine yielded an Erk-1 gene product that could not be activated. Similar results were obtained when the threonine site was mutated to valine. It is likely that the phorbol ester-stimulated protein-tyrosine/threonine kinase(s) is an up-stream target for multiple extracellular signals.
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PMID:Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product. 151 47

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of anticentromere autoantibodies in sera of patients with suspected or manifest rheumatic diseases. The antigen source used in this assay consists of the recombinant protein of glutathione S-transferase (GST) fused to the last 60 C-terminal amino acid residues of the major centromere protein CENP-B. Although this CENP-B segment is only a small part of the complete polypeptide, we show that it constitutes an important autoimmune antigenic domain which is recognized by all patient sera in which ACA can be detected using the immunoblotting technique with a HeLa S3 nuclear protein extract as antigen source.
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PMID:Molecular cloning of a major CENP-B epitope and its use for the detection of anticentromere autoantibodies. 154 84

ELP, the embryonal LTR binding protein, is a member of the nuclear receptor superfamily and a mouse homologue of Drosophila FTZ-F1. ELP is expressed specifically in undifferentiated mouse embryonal carcinoma cells and participates in suppression of the Moloney murine leukemia virus genome. The zinc finger domain of the protein was fused with glutathione S-transferase and was successfully used for isolating genomic targets. Sixteen genomic fragments were isolated and twelve of them strongly interacted with ELP. Six of the ELP binding fragments were analyzed further. All of these contained the multiple binding sites for ELP, which matched well with the consensus binding sequence for FTZ-F1, YCAAGGYCR. Among these, three fragments functioned as negative regulatory elements in response to ELP, when placed upstream to the promoter region of the Moloney leukemia virus. These results indicate that ELP may function as a negative transcription factor for a variety of cellular sequences, in addition to suppressing expression of Moloney leukemia virus in early embryonal cells. It was also shown that the procedure employed here works well for isolation of genomic targets of transcription factors.
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PMID:Isolation of high affinity cellular targets of the embryonal LTR binding protein, an undifferentiated embryonal carcinoma cell-specific repressor of Moloney leukemia virus. 157 38


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