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)

The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.
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PMID:Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli. 133 Nov 10

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

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and is required for both DNA replication and DNA repair. PCNA forms complexes with D-type cyclins, candidate G1 cyclins in mammalian cells. To better understand the functions of the complexes, we examined interactions between PCNA and D-type cyclins, using in vitro-translated mouse PCNA and mouse cyclin D1 or D3 fused to glutathione S-transferase (GST). Analysis of a set of deletion mutants of PCNA revealed that either the N-terminal (residues 2-64) or the C-terminal (residues 197-228) region is necessary for association with D-type cyclins. The cyclin binding of the chimeric protein of the N-terminal (residues 1-68) or the C-terminal (residues 195-261) region of PCNA and rat DNA polymerase beta which does not bind to the cyclins by itself supports this notion. The purified recombinant mouse PCNA expressed in Escherichia coli bound to the D-type cyclin-GST fusion proteins, thereby suggesting that PCNA binds directly to D-type cyclins, without the requirement of other cellular factors. This is apparently the first report on the structure-function relationship of PCNA which may link DNA replication and DNA repair with cell cycle control.
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PMID:D-type cyclin-binding regions of proliferating cell nuclear antigen. 790 6

A vector has been constructed to allow genetic fusions of guest antigens via a hinge domain to the C terminus of the highly immunogenic C fragment of tetanus toxin. A fusion has been constructed with the gene encoding the protective 28-kDa glutathione S-transferase (EC 2.5.1.18) from Schistosoma mansoni. The recombinant vector has been electroporated into the nonvirulent Salmonella typhimurium aroA live vaccine strain SL3261. The corresponding chimeric protein is stably expressed in a soluble form in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice immunized intravenously with a single dose of the live recombinant bacteria elicit antibodies to both fragment C and glutathione S-transferase as detected by enzyme-linked immunosorbent assays. Furthermore, all of the mice were solidly protected when challenged with lethal doses of either tetanus toxin or the virulent Salmonella typhimurium strain C5. Mice have also elicited antibodies to fragment C and glutathione S-transferase after oral immunization. It may be that a live trivalent vaccine against typhoid, tetanus, and schistosomiasis is feasible.
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PMID:Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella. 797 44

An earlier report has shown that eight viral proteins with a common amino acid sequence (R/P)RA(P/S)R are nucleotidylyated in vitro by nuclear extracts from cells infected with herpes simplex virus 1. One, the product of the alpha 22 gene, is nucleotidylylated in the absence of viral proteins made late in infection. A chimeric protein (GST22P) consisting of amino acids 50-200 of the alpha 22 coding sequence fused to the C terminus of the glutathione S-transferase was nucleotidylylated by enzymes in nuclear extracts of infected or mock-infected cells and also by a casein kinase II enzyme purified from the sea star. The enzyme did not nucleotidylylate common casein kinase II substrates (casein, phosvitin) and the reaction was inhibited by heparin. The results are consistent with the hypothesis that nucleotidylylation of the eight viral proteins involves casein kinase II.
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PMID:Casein kinase II specifically nucleotidylylates in vitro the amino acid sequence of the protein encoded by the alpha 22 gene of herpes simplex virus 1. 799 47

The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domain derived from the 3' end of gag, fused to 154 residues encoded by the 5' region of the pro open reading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted by site-directed mutagenesis to allow the read-through from the gag into the pro open reading frame. The obtained insert was then cloned into pGEX-2T, a plasmid containing the glutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30) was isolated by affinity chromatography on a glutathione-Sepharose 4B column, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparent Km for dUTP was 28 microM. The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region.
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PMID:Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity. 813 16

Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.
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PMID:Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity. 819 58

The Moloney murine leukemia virus (Mo-MuLV) protease has been cloned into the prokaryotic expression vector pGEX-2T, expressed in fusion with the glutathione S-transferase from Schistosoma japonicum, and purified to apparent homogeneity after thrombin cleavage of the chimeric protein. The purified protease showed maximum activity at pH 6.0 and was inhibited by several aspartyl protease inhibitors, found to be active toward the human immunodeficiency virus-1 (HIV-1) protease. Peptides representing maturation cleavage sites in Gag and Gag-Pol polyproteins were accurately cleaved by the recombinant protease, and kinetic parameters have been determined. In addition, oligopeptides mimicking the cleavage site found in the transmembrane protein and leading to the formation of p15E and p2E were also hydrolyzed at the expected position. The Mo-MuLV protease appears to be more closely related to the HIV-1 protease than to the mouse mammary tumor virus enzyme, based on its substrate specificity and sensitivity to aspartyl protease inhibitors.
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PMID:Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme. 837 34

Ras (Ha-Ras, Ki-Ras, N-Ras) is implicated in the regulation of various cell functions such as gene expression and cell proliferation downstream from specific extracellular signals. Here, we partially purified a Ras-interacting protein with molecular mass of about 180 kDa (p180) from bovine brain membrane extract by glutathione S-transferase (GST)-Ha-Ras affinity column chromatography. This protein bound to the GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).GST-Ha-Ras affinity column but not to those containing GDP.GST-Ha-Ras or GTP gamma S.GST-Ha-Ras with a mutation in the effector domain (Ha-RasA38). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein. The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP gamma S.GST-Ha-Ras. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP gamma S.GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.
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PMID:Identification of AF-6 and canoe as putative targets for Ras. 855 59

EWS-FLI-1 is a chimeric protein produced in most Ewing's sarcomas. It results from the fusion of the N-terminal-encoding region of the EWS gene to the C-terminal DNA-binding domain (the ETS domain) encoded by the FLI-1 ets family gene. Both EWS-FLI-1 and FLI-1 proteins function as transcription factors that bind specifically to ets sequences (the ets boxes) present in promoter elements. EWS- FLI-1 is a powerful transforming protein, whereas FLI-1 is not. In a search for potential DNA binding sites for these two proteins, we have tested their ability to recognize the serum responsive element (SRE) in the c-fos promoter. This cis element contains an ets box which can be occupied by members of the ETS protein family which do not bind DNA autonomously but form a ternary complex with a second protein, p67SRF (serum responsive factor). We demonstrate here that EWS-FLI-1, but not FLI-1, is able to form a ternary complex on the c-fos SRE. Using a GST pull-down assay, we show that both FLI-1 and EWS-FLI-1 interact in vitro with SRF in the absence of DNA. In electromobility shift assays, EWS-FLI-1 binding to the SRE is detectable in the absence of SRF whereas the binding of FLI-1 is not, suggesting that the interaction with DNA is the step which limits ternary complex formation by FLI-1. Deletion of the N-terminal portion of FLI-1 resulted in a protein which behaved as EWS-FLI-1, suggesting the existence of an N- terminal inhibitory domain in the normal protein. Taken together, our data indicate that there are intrinsic differences in the binding of EWS-FLI-1 and FLI-1 proteins to distinct ets sequences.
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PMID:SRE elements are binding sites for the fusion protein EWS-FLI-1. 860 38


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