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 core protein and the transmembrane protein, encoded for the structural genes gag and env, respectively, of caprine arthritis-encephalitis virus were amplified by use of polymerase chain reaction, cloned into a pGEX-2T vector, and expressed in Escherichia coli as fusion proteins with the glutathione S-transferase at their C-terminus. The recombinant proteins were purified and evaluated by use of an ELISA. Sera from 269 goats were tested, and the results were compared with those obtained by use of immunoblot analysis. When results from both recombinant ELISA (r-ELISA) were compared, it appeared that the transmembrane glycoprotein was more immunoreactive than the core protein, because it was recognized by a higher percentage of sera from infected goats. When results of the 2 ELISA (p28 r-ELISA and p40 r-ELISA) were combined in parallel, they were comparable to those of the immunoblot test, with sensitivity of 100% and specificity of 98.3%. It was also found that use of both r-ELISA makes it possible to compare the variable immunoreactivity against gag and env viral antigens, which may be correlated with the disease state. The r-ELISA, using core and transmembrane proteins, appears to be highly sensitive and specific for detection of antibodies against caprine arthritis-encephalitis virus.
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PMID:Bacterial expression of the caprine arthritis-encephalitis virus gag and env proteins and their use in enzyme-linked immunosorbent assay. 757 48

PG-M is a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds. We previously isolated cDNA clones encoding the core protein of PG-M (Shinomura, T., Nishida, Y., Ito, K., and Kimata, K. (1993) J. Biol. Chem. 268, 14461-14469). The amino acid sequence deduced from the cDNA analysis revealed the presence of two epidermal growth factor-like domains, a C-type lectin-like domain, and a complement regulatory protein (CRP)-like domain at the COOH terminus. The COOH-terminal portion has been expressed as a fusion protein with glutathione S-transferase in Escherichia coli to test its carbohydrate binding activity using affinity chromatography. The purified fusion protein binds to immobilized D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine in a calcium-dependent manner. Furthermore, the fusion protein binds to heparin- or heparan sulfate-Sepharose. To investigate roles of each COOH-terminal domain, we have made a truncated construct which lacks the CRP-like domain and determined if the CRP-like domain is involved in the binding activity. The removal of this domain resulted in the complete loss of both C-type lectin-like and heparin binding activities. The results suggest that a whole set of epidermal growth factor-, lectin-, and CRP-like domains may serve a functional structure for these bindings.
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PMID:Expression and binding activity of the carboxyl-terminal portion of the core protein of PG-M, a large chondroitin sulfate proteoglycan. 796 77

Hepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and may possess multiple functions. In this study, we demonstrated the homotypic interaction and multimerization of HCV core protein in vitro and in vivo. By using a yeast two-hybrid system, we showed that the amino-terminal hydrophilic portion (amino acids 1-115) of the core protein could interact with itself. Deletion analysis mapped the interacting domain within amino acid residues 36-91. The homotypic interaction of the core protein was also confirmed by in vitro protein-protein blotting assay using the recombinant HCV core proteins and by its binding to the glutathione S-transferase core fusion protein. The biological significance of the core protein self-interactions was demonstrated by the detection of multimeric forms of the core protein in mammalian cells. The domain responsible for multimerization was determined to be within the amino-terminal hydrophilic region (amino acids 1-115). Both the membrane-bound and the free core proteins exist in dimeric and multimeric forms, suggesting that multimerization of the HCV core protein occurred at an early stage of viral assembly and that the multimer forms may be involved in multiple functions of the core protein.
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PMID:Homotypic interaction and multimerization of hepatitis C virus core protein. 861 40

A full-length and a truncated gene for the core protein of hepatitis C virus (HCV) were linked to the gene for glutathione S-transferase (GST), and the expression of each GST-HCV core fusion protein was analyzed. The truncated GST-HCV core (1-123) fusion protein was expressed as a mostly soluble and partly insoluble form comprising more than 50% of the total protein in Escherichia coli after induction by isopropylthio-beta-D-galactoside (IPTG), while the full length GST-HCV core (1-191) fusion protein was not expressed, suggesting that the hydrophobic carboxy terminal region in the core protein affects its expression. In addition, the GST-HCV core (1-123) fusion protein purified by GST-agarose chromatography reacted specifically with an anti-HCV serum from a patient.
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PMID:Overexpression and simple purification of a truncated, immunologically reactive GST-HCV core (1-123) fusion protein. 879 26

Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.
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PMID:High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice. 882 96

Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth. In this study we investigated the known binding of decorin to human collagen XIV. This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase. Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase. Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV. In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.
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PMID:Localization of a binding site for the proteoglycan decorin on collagen XIV (undulin). 925 49

An established mouse model system was used to evaluate the effectiveness of the major outer core protein VP7 of African horse sickness virus (AHSV) serotype 9 as a subunit vaccine. Balb C mice were immunised with VP7 crystals purified from AHSV infected BHK cells. In groups of mice, each of which was immunised with > or = 1.5 micrograms of the protein in Freund's adjuvant, > or = 80% of mice survived challenge with a virulent strain of a heterologous AHSV serotype (AHSV 7), that killed > or = 80% of the mice in the uninoculated control groups. This level of protection was significantly greater than that observed in mice inoculated with equivalent amounts of either denatured VP7 (50% survival), or GST/VP7 fusion protein (50-70% survival), or which were vaccinated with AHSV 9 (40-50% survival). The VP7 protein folding, or its assembly into crystals, are thought to play some role in the effectiveness of the protective response observed. Titres of circulating antibodies against AHSV VP7 were determined by competitive ELISA but did not appear to correlate with the levels of protection observed. Passive transfer of these antibodies to syngeneic recipients also failed to protect Balb C mice from the AHSV 7 challenge. The observed protection is therefore unlikely to be due to an antibody mediated immune response.
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PMID:VP7 from African horse sickness virus serotype 9 protects mice against a lethal, heterologous serotype challenge. 978 8

In addition to being a structural protein that packages the viral genomic RNA, hepatitis C virus (HCV) core protein possesses regulatory functions. In this report, we demonstrate that the HCV core protein could enhance the gene transactivation activity of the tumor suppressor p53, regardless of whether p53 was derived from an exogenous or an endogenous gene. The activation of p53 by the HCV core protein was supported by the observation that the HCV core protein could enhance the expression of p21(waf1/Cip1), a downstream effector gene of p53, in a p53-dependent manner. Further studies indicated that the HCV core protein could also suppress hepatocellular growth via p53. The HCV core protein and p53 could bind to each other in vitro, which was evidenced by the coimmunoprecipitation, the GST pull-down, and the Far-Western blot assays. The deletion-mapping analysis indicated that the carboxy-terminal sequence of p53 located between amino acids 366 and 380 was required for the core protein binding. These results raised the possibility that the HCV core protein might activate p53 through direct physical interaction. The persistent perturbation of p53 activity by the HCV core protein during chronic infection may have important consequences in HCV pathogenesis.
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PMID:Activation of p53 tumor suppressor by hepatitis C virus core protein. 1054 38

The core protein (Core) of hepatitis C virus (HCV) has been known to play an important role in hepatocarcinogenesis. By using glutathione S-transferase (GST) pull-down assay, we show here that Core formed a complex with p21Waf1/Cip1/Sdi1 (p21) cell cycle regulator. The deletion-mapping analysis revealed that a portion near the N-terminus of Core (amino acids 24-52) and a C-terminal portion of p21 (amino acids 139-164) were involved in the complex formation. The complex formation was not impaired by point mutations of p21 at residues 147, 149, and 150, which have been reported to abrogate interaction of p21 with proliferating cell nuclear antigen (PCNA), discriminating the Core-binding sequence from the PCNA-binding sequence. Due to the close vicinity of the binding sites, however, Core and PCNA competed with each other when interacting with p21. The distinct interaction between Core and p21 may provide a new aspect to the studies of HCV pathogenesis.
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PMID:Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1. 1087 31

The abnormal appearance and age-dependent loss of resident fibroblast growth factor receptor-2 (FGFR2) and gain of activity of FGFR1 in epithelial cells is a hallmark of the slow progression to malignancy in some models of prostate cancer. Pericellular matrix heparan sulfate (HS) is an integral subunit of the FGFR tyrosine kinase complex that restricts activity in absence of FGF, facilitates binding of an activating FGF, and confers specificity for FGF isoforms. In this report, we isolated and purified HS proteoglycan (HSPG) from premalignant prostate tumor epithelial cells based on the ability of the HS chains to form a binary complex with immunoglobulin module II of the ectopic and progression-promoting FGFR1 that was competent to bind FGF. The FGFR1 affinity-purified product exhibited a specific activity of over 600 times that of crude cellular HSPG enriched from cell lysates by ion exchange chromatography. The purified preparation exhibited a single NH(2)-terminal sequence with 11 of 13 residues identical to syndecan-1. The activity of purified recombinant glutathione S-transferase-tagged syndecan-1 expressed in premalignant epithelial cells confirmed that syndecan-1 bears HS chains that exhibit the rare motif that forms the FGF-binding complex with ectopic FGFR1. These results are the first to identify by affinity purification a specific HSPG core protein, the HS chains of which act as an integral subunit of the FGFR complex. The results suggest that syndecan-1 provides HS chains in premalignant epithelial cells to both the FGFR2- and FGFR1-signaling complexes that are integral to their dual roles in progression to malignancy.
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PMID:A rare premalignant prostate tumor epithelial cell syndecan-1 forms a fibroblast growth factor-binding complex with progression-promoting ectopic fibroblast growth factor receptor 1. 1143 73


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