Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An expression vector yielding large amounts of GST P1-1 in the cytoplasm of Escherichia coli was constructed. The recombinant enzyme, obtained after purification, was characterized in its physicochemical and kinetic properties and appeared to be indistinguishable from that purified from human placenta. However, N-terminal amino acid sequencing revealed that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not simply due to overproduction of GST. In fact, under growth conditions that lead to a sharp decrease in the production of the protein the N-terminal methionine was not removed. GST was unable to translocate across the bacterial membrane when it was fused to the leader peptide of the pelB gene from Erwinia carotovora and accumulated in the cytoplasm in a soluble and active conformation. However, when this fusion protein was produced in a bacterial strain overexpressing the bacterial chaperonins GroEL and GroES, a fraction of GST was exported into the periplasmic space with the correct N-terminal sequence. The yield of correctly processed GST accounted for 12% of total GST present in the E. coli cells. Our results suggest that chaperonins are able to interact with nascent GST, thus maintaining the protein in an export-competent form and that E. coli strains with enhanced secretory characteristics may be obtained by genetic engineering technology.
 ...
PMID:Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli. 853 49

Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum. The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies. The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity. The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C. The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES. The catalytic properties of a recombinant C. thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E. coli [Ciruela (1995) Ph.D. Thesis, University of Kent] were identical with those of the authentic C. thermocellum Cpn60. Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer. Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E. coli GroEL complexes.
 ...
PMID:Thermostable chaperonin from Clostridium thermocellum. 868 8

The groESL operon from Clostridium thermocellum (Ct) has been isolated and sequenced, revealing two ORFs of 285 and 1626 nt, separated by 48 nt. The first ORF encoded a 94-aa 10.6-kDa GroES homologue; the second encoded a 541-aa polypeptide of 57.6 kDa, that exhibited 61% and 77% sequence identity with GroEL from Escherichia coli (Ec) and Clostridium acetobutylicum (Ca), respectively. A putative tsp, preceded by -10 and -35 consensus promoters, was identified upstream of groES. This was followed by an inverted repeat observed previously in bacterial heat shock genes. A 15-nt palindrome characteristic of a Rho-independent transcription terminator, was located downstream of groEL. The first nt of the groES translational start codon was preceded (7 nt) by a putative RBS (AGGAGG); a second RBS sequence was located 8 nt upstream of the groEL start. Production of GroE homologues by Ct was constitutive, but was enhanced significantly during a temperature upshift from 60 degrees C to 70 degrees C. The Ct GroEL, expressed in Ec as a fusion protein with GST, was purified, free of contaminating Ec GroEL.
 ...
PMID:Sequence and transcriptional analysis of groES and groEL genes from the thermophilic bacterium Clostridium thermocellum. 904 57

Steroid hormone receptors (SHR) form complexes with heat shock proteins (hsps). The 1alpha,25-dihydroxyvitamin D(3) receptor (VDR) has not been previously shown to interact with hsps. During expression and purification of VDR-glutathione S-transferase (VDR-GST) fusion proteins encompassing full-length, DNA, and ligand-binding domains of the VDR (FL-VDR, DBD-VDR, and LBD-VDR), we observed binding of bacterial hsps with VDR-GST constructs. All VDR constructs bound DnaK in amounts greater than GST alone and bound smaller amounts of DnaJ or GrpE. GroEL bound only to FL-VDR. GroES did not bind to VDR. When VDR-GST constructs were incubated with a reticulocyte lysate system that has been used previously to examine SHR-hsp interactions, eukaryotic hsc70 was detected bound to FL-VDR and DBD-VDR. Binding of hsp90 to VDR was not detected. However, geldanamycin, an hsp90 inhibitor, reduced 1alpha,25-dihydroxyvitamin D(3)-mediated gene activation in osteoblasts. Our data show that the bacterial and eukaryotic hsps associate with the VDR and might be involved in VDR function.
 ...
PMID:Association of prokaryotic and eukaryotic chaperone proteins with the human 1alpha,25-dihydroxyvitamin D(3) receptor. 1040 88

According to codon preference of Escherichia coli, the optimized coding sequence of human vasostatin120-180aa (VAS) was obtained by chemical synthesis and molecular cloning methods. Using PCR and enzyme digestion, the full encoding sequence for VAS was cloned into the E. coli expression vector pALEX and expressed as a GST fusion protein in BL21 (DE3) strain. GST-VAS protein approximately accounted for 45% of the total bacterial proteins. Most of target protein existed in inclusion body. To improve the solubility of GST-VAS, the contribution of low temperature and molecular chaperone co-expression to the solubility of GST-VAS was tested. The results showed that co-expression with chaperons, TF and GroES/GroEL, and low expression temperature cooperatively improved the solubility of GST-VAS from 10 to 85%, and the yield of soluble GST-VAS was sixfold increased. When purified by GST affinity chromatography, 50 mg GST-VAS was obtained with purity over 85% from 1 L culture. Intact VAS was released by enterokinase digestion and further purified by Sephadex G50 gel filtration chromatography. About 7.2 mg intact homogeneous VAS protein was finally produced from 1L bacterial culture. The identity of GST-VAS and VAS was validated by Western blotting analysis. Recombinant VAS protein displayed distinct inhibition of endothelial cell proliferation and anti-angiogenic activity by chick embryo chorioallantoic membrane assay.
 ...
PMID:Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization. 1564 81

It was shown that the chaperonin GroEL/GroES and protease Lon influence the expression of the Vibrio fischeri lux regulon in Escherichia coli cells: E. coli groE mutants bearing hybrid plasmid with the lux regulon were weakly luminescent; cells of the E. coli lon- comprising the entire lux regulon display very intense bioluminescence, with no lag period in the induction curve characteristic of lon+ strains. The luxR gene was cloned from the Vibrio fischeri genome in the pGEX-KG vector. It was shown that the active fusion protein GST-LuxR by affinity chromatography on glutathione-sucrose colony is purified only with proteins GroEL and Lon. The present results showed that the LuxR, transcriptional activator of the V. fischeri lux operon, really complexes with GroEL chaperonin and Lon protease. We suppose, that the GroEL/GroES chaperonin systems is required for the folding of LuxR into an active protein, and the LuxR is the target for the ATP-dependent serine Lon protease of E. coli.
 ...
PMID:[Role of GroEL/GroES chaperonin system and Lon protease in regulation of expression Vibrio fischeri lux genes in Escherichia coli cells]. 1663 68

It has been shown that the chaperonin GroEL, together with GroES co-chaperonin and Lon ATP-dependent protease are involved in the regulation of expression of the Vibrio fischeri lux operon in Escherichia coli cells. The cells of E. coli groE (pF1)- bearing a plasmid with the complete V. fischeri lux regulon were weakly luminescent. The cells of E. coli lonA (pF1) displayed intense bioluminescence. The same effects also occurred in mutant E. coli strains bearing a hybrid plasmid pVFR1, where the luxR gene and the regulatory region of the V. fischeri lux operon were inserted before the Photorhabdus luminescens luxCDABE cassette. The V. fischeri luxR gene was cloned in the pGEX-KG vector with the formation of a hybrid gene gst-luxR. It was shown that affinity chromatography of the product of expression, the chimeric protein GST-LuxR, on a column with glutathione-agarose resulted in its copurification with the proteins GroEL and Lon. Consequently, LuxR, the transcription activator of the lux operon, forms complexes with these proteins. It is supposed that GroEL/GroES is responsible for the folding of the LuxR protein, and Lon protease degrades the LuxR protein either before its folding into an active globule or at denaturing.
 ...
PMID:[Host factors in the regulation of the Vibrio fischeri lux operon in Escherichia coli cells]. 1702 79

In Brassicaceae, a self-incompatibility (SI) system mediates pollen-pistil interactions. Self-pollen could be recognized and rejected by incompatible pistils. Several components involved in the SI response have been determined, including S-locus receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11, and arm repeat-containing protein 1 (ARC1). However, the components involved in the SI system of Brassicaceae are not fully understood. Here, we detected expression patterns of 24 SI-related genes in non-heading Chinese cabbage (Brassica campestris ssp chinensis Makino) after compatible and incompatible pollination, and potential interaction relationships of these genes were predicted. SRK and ARC1 transcripts increased initially 0.25 h after incompatible pollination, while kinase-associated protein phosphatase had an expression pattern that was opposite that of SRK transcripts during self-pollination. Plant U-box 8 was not required in the SI response of non-heading Chinese cabbage. Our results showed that the SI signal of non-heading Chinese cabbage could occur within 0.25 h after self-pollination. The hypothetical interaction relationships indicated that plastid-lipid-associated protein and malate dehydrogenase could be negatively regulated by chaperonin 10, glutathione transferase, cytidylate kinase/uridylate kinase, and methionine synthase by indirect interactions. Our findings could be helpful to better understand potential roles of these components in the SI system of non-heading Chinese cabbage.
...
PMID:Expression analysis of self-incompatibility-associated genes in non-heading Chinese cabbage. 2506 91

Deformed wing virus (DWV) interacting with Varroa destructor is a possible cause of honeybee colony mortality. VP2 is the structural protein of DWV but its function remains unknown. To clarify the function of VP2 and screen for novel binding proteins that interact with VP2, we carried out a membrane protein yeast two-hybrid screening using VP2 as bait. Subsequently, the interaction between VP2 and the host interacting protein [heat shock protein 10 (Hsp10)] was further verified using glutathione S-transferase pull-down assay in vitro and co-immunoprecipitation assay in cells. Furthermore, fluorescence confocal microscopy revealed that VP2 and Hsp10 were mainly co-localized in the cytoplasm. Using real-time polymerase chain reaction, we found that Hsp10 expression in DWV-infected worker honey bees were downregulated compared with that in healthy honey bees. Additionally, we showed that overexpression of VP2 protein could reduce the expression of Hsp10. These results suggest that Hsp10 plays a vital role in host immunity and antiviral effects.
 ...
PMID:Identification of a novel host protein interacting with the structural protein VP2 of deformed wing virus by yeast two-hybrid screening. 3265 7