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)

The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2. We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein beta-lactamase is Pas7p dependent. In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro. For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient. The specific binding of Pas7p to thiolase does not require peroxisomes. Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e. the activation domain of Gal4p or GST. These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2. Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired.
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PMID:The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene. 867 Jul 91

The gene from Bacteroides fragilis encoding a metallo-beta-lactamase, ccrA, was expressed in Escherichia coli BL21(DE3) containing the wild-type disulfide bond-catalyzing system dsb as an active, soluble enzyme in quantities exceeding 100 mg/liter using both rich and minimal media. Both the nonfusion and a glutathione S-transferase fusion enzyme lacking the periplasmic signal sequence were purified to homogeneity. Characteristics of the purified nonfusion enzyme are shown to be similar to those of the renatured enzyme previously reported. Thermal denaturation studies using circular dichroism and fluorescence spectroscopy show that CcrA undergoes a transition at approximately 50 degrees C which corresponds to the transition temperature of catalytic activity. The secondary structure of the protein and the catalytic apparatus are thus intimately linked.
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PMID:High-yield expression, purification, and characterization of active, soluble Bacteroides fragilis metallo-beta-lactamase, CcrA. 912 7

The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone did not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S. lividans. The latter clone contains two open reading frames, blaA and blaB, located just upstream of but transcribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL. The deduced BlaA protein belongs to the LysR family of transcription regulators. In order to examine the role of BlaA in regulation, we here report on over-expression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region. The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N11-A motif, a characteristic of LysR-type promoters. Another T-N11-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N11-A motifs in BlaA binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S. lividans host the capacity to produce inducible beta-lactamase. It can thus be concluded that the S. cacaoi blaL and blaU genes are controlled by the same regulatory system.
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PMID:The two beta-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system. 923 76

In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features. This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e. electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption). The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries. Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles. Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries. This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments.
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PMID:Using a residue clash map to functionally characterize protein recombination hybrids. 1498 83

Random oligonucleotide fragments were designed and amplified by PCR and fused with the activating domain of pGAD424 to construct a random peptide library. The DNA fragment encoding beta-lactamase was fused with the binding domain of pGBT9(+2). Subsequently, using yeast two-hybrid system we found two positive clones encoding peptides P1 and P2 that have the ability to bind beta-lactamase in vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed in Escherichia coli and isolated by glutathione-Sepharose 4B affinity chromatography. Finally, P1 and P2 were cleaved from the fusion protein with thrombin and purified by ultrafiltration. Inhibition assay of peptides with beta-lactamase in vitro indicated that only P1 has the ability to inhibit beta-lactamase.
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PMID:Identification of beta-lactamase inhibitory peptide using yeast two-hybrid system. 1609 38

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.
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PMID:Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli. 1620 24

We evaluated the in vitro activity of fosfomycin against a total of 192 CTX-M beta-lactamase-producing Escherichia coli strains isolated in 70 Japanese clinical settings. Most of the isolates (96.4%) were found to be susceptible to fosfomycin. On the other hand, some of the resistant isolates were confirmed to harbor the novel transferable fosfomycin resistance determinants named FosA3 and FosC2, which efficaciously inactivate fosfomycin through glutathione S-transferase activity.
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PMID:Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes. 2040 16

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate, Helixate NexGen and Refacto), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refacto showed glutathione S-transferase and beta-lactamase, whereas Advate apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants.
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PMID:Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation. 2073 44