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)

Sequences encoding the 27K and 25K nef gene products (Nef 27 and Nef 25) were amplified by PCR from a human immunodeficiency virus type 1 infectious clone and subcloned directly into Escherichia coli, yeast and baculovirus expression vectors. The yeast- and baculovirus-derived Nef had native N termini but the expression levels were low. The expression levels of the E. coli-derived glutathione S-transferase-Nef fusion proteins were very high and a major portion was soluble. Large-scale production of E. coli-derived Nef 27 and Nef 25 was carried out by growing recombinant cells in a fermenter under fed-batch conditions followed by affinity purification on glutathione-Sepharose before and after thrombin cleavage. Large quantities of highly purified recombinant Nef proteins have been produced for functional and structural studies. Under non-reducing conditions both Nef 27 and Nef 25 existed as a mixture of monomers, dimers and small amounts of higher oligomers, but when reduced were monomeric. The highly purified Nef proteins had no G protein activities, however Nef 27 was biologically active. When electroporated into uninfected CD4+ T lymphocytes both E. coli-derived Nef 27 and yeast-derived myristylated Nef 27 down-regulated the surface expression of CD4, demonstrating that this method can be used to assess the biological activity of purified recombinant Nef.
J Gen Virol 1994 Mar
PMID:Large-scale production and characterization of recombinant human immunodeficiency virus type 1 Nef. 812 63

Transcription of a putative mitochondrial gene (orf138) has previously been correlated with Ogura cytoplasmic male-sterility (CMS) in rapeseed cybrids. In this paper, studies performed on a Brassica cybrid with a different organization of the orf138 locus confirm this association. We also show that mitochondria isolated from male-sterile rapeseed plants synthesize a polypeptide of 19 kDa, which is absent in fertile revertants. Antibodies against a glutathione S-transferase-ORF138 fusion protein were raised to establish that this 19 kDa polypeptide is the product of orf138. The anti-ORF138 serum was used to demonstrate that the orf138 translation product occurs only in sterile cybrids and co-purifies with the mitochondrial membrane fraction.
Mol Gen Genet 1994 Jun 03
PMID:Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids. 820 45

The transcription factor ALCR of the ethanol utilisation pathway in Aspergillus nidulans contains a zinc binuclear motif (CysX2CysX6CysX16CysX2CysX6Cys), within the DNA-binding domain located in the N-terminal region of the ALCR protein. Specific targets have been localised in the promoter of the alcR gene, involved in the autoregulation process, and in the promoter of the structural gene alcA (encoding alcohol dehydrogenase I), which is also under the control of ALCR. The DNA-binding domain has been expressed in-Escherichia coli as a GST-ALCR (7-58*) fusion protein and also obtained as an ALCR (7-58*) peptide. Both the ALCR fusion protein and the ALCR peptide are able to bind 65Zn(II) in vitro, if reduction of cysteines occurs prior to the addition of zinc. Competition experiments showed that Cd(II), Co(II) and Cu(II) are efficient competitors for the zinc binding sites. The ALCR DNA-binding domain was shown to contain 2 mol of tightly bound Zn(II) per mole of fusion protein. Removal of the intrinsic Zn(II) requires treatment with Chelex. This treatment abolishes the ability of the protein to bind to the targets of ALCR located in the alcA and alcR promoters. The apo-ALCR DNA-binding motif could be reconstituted with Zn(II) or Cd(II), restoring specific DNA binding to both types of targets. Thus a direct relationship was shown to exist between the zinc content of ALCR and its DNA-binding activity.
Mol Gen Genet 1994 Jan
PMID:Relationship between zinc content and DNA-binding activity of the DNA-binding motif of the transcription factor ALCR in Aspergillus nidulans. 827 45

Full-sized gen vif of human immunodeficiency virus has been synthesized and cloned into plasmid pGEX-2T. Vif-gene expression was found in Escherichia coli cells resulting in production of a hybrid GST-protein. The recombinant protein studied by the immunoblotting technique reacted with 8 of 22 probes of human HIV-positive sera. The recombinant protein is specifically cut by thrombin in two proteins corresponding to GST and VIF-proteins in molecular mass.
Mol Gen Mikrobiol Virusol
PMID:[Expression of the vif gene of human immunodeficiency virus type 1 in Escherichia coli and study of the immunoreactivity of the vif protein]. 828 43

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the overproduced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.
Mol Gen Genet 1993 Jul
PMID:Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans. 839 15

The first predicted polypeptide encoded by the potyvirus tobacco vein mottling virus (TVMV) is a highly positively charged protein of predicted M(r) 29K that functions as a protease to perform the first predicted cleavage in the potyvirus polyprotein. We expressed this protein (P1pro) fused with glutathione S-transferase (GST) and purified the fusion protein from engineered Escherichia coli. We found that the intact fusion protein, as well as samples in which the P1pro portion was liberated from GST by pretreatment with thrombin, was able to bind RNA. Binding activity was optimal at relatively high KCl concentrations, suggesting an interaction dependent on a specific protein structure and not just on the binding of the negatively charged phosphate backbone by the positively charged P1pro polypeptide. The TVMV P1pro preferred ssRNA over DNA or dsRNA, and showed a possible preference for sequences containing oligo(G) tracts. Like other potyvirus-encoded proteins, the TVMV P1pro therefore possesses more than one demonstrable biochemical activity and probably plays multiple roles in the TVMV life cycle.
J Gen Virol 1993 Jun
PMID:The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein. 850 65

An analysis of linear antibody-binding sites of the glycoprotein B (gB) molecule of murine cytomegalovirus (MCMV) and of genetic variation within these regions was performed. To achieve this, a series of overlapping fragments spanning the entire coding sequence of the gB gene of the K181 strain of MCMV was expressed in E. coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system. Four antibody-binding regions were mapped to locations spanning amino acid residues 17-79 (BS), 155-278 (BE2), 809-926 (SS) and 347-508 (BB and EE), based on reactivity in Western blot analysis of GST-gB fusion proteins with murine polyclonal antiserum raised against MCMV. Only the antibody-binding region BE2 (155-278) elicited an antiserum that exhibited complement-dependent neutralizing activity, and immunization of mice with the fusion protein BE2 led to moderate but significant reductions in the level of MCMV replication in the spleen. Polyclonal antisera raised against the GST-gB fusion proteins detected purified virion proteins of 105 kDa (anti-BS and anti-BE2) and 52 kDa (anti-SS) and are therefore likely to recognize the N-terminal and C-terminal portions of the gB molecule, respectively. The antibody-binding region within amino acid residues 17-79 was found to be MCMV strain-specific, whereas antibody-binding regions within residues 155-278 and 809-926 were found to be conserved among MCMV field isolates. Comparative sequence analysis of the corresponding regions of MCMV gB revealed a level and extent of sequence of sequence heterogeneity consistent with these findings.
J Gen Virol 1996 Jan
PMID:Assessment of antigenicity and genetic variation of glycoprotein B of murine cytomegalovirus. 855 28

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 of Schizosaccharomyces pombe was expressed in Escherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with 32P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutant rpb2 alleles, rpb2E357A and rpb2H386L, each carrying a single amino acid substitution at the site corresponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutant rpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutant rpb2 alleles were expressed in an rpb2-disrupted S. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.
Mol Gen Genet 1996 Jan 15
PMID:Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II. 856 79

The barley stripe mosaic virus (BSMV) gamma-b gene encodes a 17 kDa cysteine-rich protein known to affect virulence and to have a role in regulating viral gene expression. We have constructed recombinant gamma-b-glutathione S-transferase fusion proteins in Escherichia coli and have determined the ability of the purified fusion proteins and various mutant derivatives to bind nucleic acids in vitro. Gel-shift analyses revealed that the wild-type gamma-b-fusion protein is able to bind RNA cooperatively. The binding affinity is highly selective for single-stranded RNA because double-stranded RNA, single-stranded and double-stranded DNA, and transfer RNA were unable to compete for binding with the labelled RNA probes. However, BSMV-specific sequence binding was not observed since a chloroplast RNA competed for binding with 32P-labelled transcripts derived from the BSMV genome. The first 44 amino acids of the 152 amino acid gamma-b fusion protein encompassing one of two cysteine-rich 'zinc finger-like' motifs, and a basic region separating the finger-like motifs are required for RNA binding. Site- specific amino acid substitutions within two groups of lysine and arginine residues located in the basic motif reduced the binding affinity of the fusion protein greatly, but cysteine and histidine substitutions designed to disrupt the finger-like motifs failed to have appreciable effects on binding. These findings indicate that the regulatory properties of gamma-b may be mediated in part by RNA binding activities.
J Gen Virol 1996 May
PMID:RNA-binding activities of barley stripe mosaic virus gamma b fusion proteins. 860 84

The African swine fever virus (ASFV) open reading frame (ORF) that is named jl8L in the Malawi (LIL20/1) isolate and E199L in the Ba71V isolate encodes a cysteine rich protein of 195 amino acids with a predicted molecular mass of 21.7 kDa and a hydrophobic domain near the C terminus. There are several possible motifs for glycosylation, phosphorylation and myristoylation. Rabbit antisera and monoclonal antibodies raised against a recombinant ASFV j18L protein expressed as a fusion protein with glutathione S-transferase (GST) identified proteins of 19.0-20 kDa in cells infected with different ASFV strains and with a recombinant vaccinia virus expressing j18L. The monoclonal antibodies detected a protein of 20.0 kDa whereas rabbit antisera detected two proteins with relative molecular masses of 15.0 and 20.0 kDa in purified extracellular ASF virions. In ASFV-infected cells, the j18L protein was expressed late post-infection and was localized mainly in the viral factories.
J Gen Virol 1996 May
PMID:Characterization of the African swine fever virion protein j18L. 860 90


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