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 epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.
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PMID:The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli. 144 91

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 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.
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PMID:Inter-species variation of schistosome 28-kDa glutathione S-transferases. 151 33

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.
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PMID:Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni. 169 15

A 183-bp fragment encoding variable domain IV (VD IV) of Chlamydia trachomatis serovar B major outer membrane protein (MOMP) (amino acids 273 to 333) and containing the species-specific epitope was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-VD IV). The fusion protein was affinity purified under nondenaturing conditions and used to immunize rabbits. Antisera were characterized by microimmunofluorescence, immunoblot, dot blot, peptide enzyme-linked immunosorbent, and in vitro neutralization assays. Antisera recognized MOMP from all 12 tested serovars of C. trachomatis but not from Chlamydia psittaci. In a dot blot assay, antisera bound to elementary bodies of serovars B, D, E, L2, and K in a strong fashion and to elementary bodies of serovars F, G, A, and H in a weak fashion but not to elementary bodies of serovars C, J, and I. High-resolution peptide mapping with synthetic overlapping serovar B MOMP peptides in a solid-phase enzyme-linked immunosorbent assay showed that immunization with GST-VD IV produced a serologic response that closely mimicked the response produced with purified serovar B elementary bodies. Antipeptide antibodies with strong binding to species- and subspecies-specific epitopes were elicited. Antisera were able to neutralize only those C. trachomatis serovars that bound antibodies in the dot blot assay. These results suggest that antigenic fragments from VD IV containing the species-specific epitope may be useful in the construction of a chlamydial vaccine for some but not all C. trachomatis serovars.
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PMID:Immunologic characterization of a cloned fragment containing the species-specific epitope from the major outer membrane protein of Chlamydia trachomatis. 170 15

Studies over the past 20 years have clearly shown the potential for developing vaccines against larval cestode infections of man and animals. The important larval cestode infections of man (Echinococcus granulosus--hydatidosis: Taenia solium--cysticercosis) involve domesticated animals as intermediate hosts in their natural life-cycles. These animals develop strong immunity against reinfection, and immunity can be artificially induced by vaccination with oncosphere antigens. A major stumbling block in developing commercial vaccines against cestodes has been the difficulty in obtaining adequate supplies of these antigens. Recent studies with Taenia ovis, a larval cestode causing cysticercosis in sheep, have demonstrated the feasibility of developing commercial vaccines against cestodes using recombinant DNA technology. A cDNA library prepared using mRNA obtained from T. ovis oncospheres was used to isolate a clone which expressed T. ovis polypeptide antigen 45W as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-45W). GST-45W gave up to 94% protection against challenge infection when used to vaccinate sheep with saponin as adjuvant. The vaccine antigen was shown by SDS PAGE to be unstable, a major disadvantage in subsequent attempts to obtain high yields of antigen for commercial production. The fusion protein has now been stabilized by reducing the size of GST-45W cDNA through deleting 19 carboxyl terminal hydropathic acids, and the resultant fusion protein GST-45W (B/X) was highly host-protective. Another experiment showed that the 45W T. ovis polypeptide cleaved enzymatically from GST-45W was still host-protective, suggesting that GST had no influence on the immunogenicity of GST-45W fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cestode vaccines. 182 8

Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets. From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated. Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families. Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction. The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum. Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins. All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera. Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T. taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively. Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.
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PMID:Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response. 182 41

Two monoclonal antibodies have been produced that bind to separate epitopes on the Mr 26,000 glutathione S-transferase (GST) of Schistosoma japonicum worms (Sj26). Both antibodies have been used in an enzyme immunoassay (EIA) with sera from infected individuals from the Philippines. Relatively high signals were obtained with sera from some, but not all, individuals who are positive for fecal eggs. Evidence was obtained that the material detected by the monoclonal antibodies was present in minute amounts and in some sera was bound in a complex with phosphorylcholine-containing molecules. It could not be absorbed by reaction with glutathione-agarose columns. There was no detectable immunoglobulin in the complex. The possibility exists that the complexes are composed of schistosome GST, or fragments, and damaged tegumental lipids shed as a result of surface immune attack. However, the presence of the native Sj26 molecule has not been proven. More detailed longitudinal studies in endemic areas are required to determine whether the assay can be used as an indicator of acquired resistance ("concomitant immunity") and whether it will be useful in the search for immunological correlates of this resistance in humans.
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PMID:Schistosoma japonicum: monoclonal antibodies to the Mr 26,000 schistosome glutathione S-transferase (Sj26) in an assay for circulating antigen in infected individuals. 210 94

Rat renin fused at the N-terminus with Sj26, a 26,000 Da glutathione S-transferase of Schistosoma japonicum, was expressed in Escherichia coli. The fusion protein was soluble and easily purified from crude bacterial lysates by affinity chromatography on immobilised glutathione. The fusion protein possessed no detectable renin activity. Antisera raised in rabbits against the fusion protein were specific for renin. These antisera did not bind soluble renin but bound immobilized renin. By immunoblotting, these antisera demonstrated rat renin to migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two broad bands of 33,000-34,000 and 35,000-37,000 Da. By immunocytochemistry of rat tissues, these antisera stained renin containing cells in the afferent arteriole of the glomerulus of the kidney, the zona glomerulosa of the adrenal and the corpus luteum of the ovary. However, apart from the afferent arteriole of the kidney, no immunoreactive renin was identified in blood vessels of the kidney, adrenal or ovary. These studies demonstrate that a recombinant renin fusion protein is a valuable alternative approach for the preparation of renin-specific antisera.
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PMID:Production of rat renin fusion protein in Escherichia coli and the preparation of renin-specific antisera. 226 96


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