Search results for "Heterologous expression."
showing 10 items of 48 documents
Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically invol…
2006
Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 A, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 A.
Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells.
2003
We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation o…
Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems
1998
Abstract Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules leading to electrophilic cations which may form adducts with DNA and other important cellular structures. Since endogenous sulfotransferases do not appear to be expressed in indicator cells of standard mutagenicity tests, rat and human sulfotransferases have been stably expressed in his−Salmonella typhimurium strain TA1538 and Chinese hamster V79 cells. Using these recombinant indicator cells, sulfotransferase-dependent genotoxic activities were detected…
Expression of human estrogen sulfotransferase in Salmonella typhimurium: differences between hHST and hEST in the enantioselective activation of 1-hy…
1998
Various human sulfotransferases (hP-PST, hM-PST, hHST) and rat sulfotransferases (rPST-IV, rHSTa) have already been expressed in Ames' Salmonella strains (in particular in TA1538). Now a further strain, TA1538-hEST, which expresses the human estrogen sulfotransferase (hEST), has been constructed. This strain activated the primary benzylic alcohol 1-hydroxymethylpyrene (1-HMP) and the secondary benzylic alcohol 1-hydroxyethylpyrene (1-HEP) to mutagens. Human sulfotransferases hEST and hHST both activated 1-HEP, but they differed substantially in their enantioselectivity for this compound.
Yeast vectors for the integration/expression of any sequence at theTYR1 locus
2007
We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vector's selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, s…
Improved acid tolerance of a recombinant strain of Escherichia coli expressing genes from the acidophilic bacterium Oenococcus oeni.
2001
Aims:Oenococcus oeni is a lactic acid bacterium used in wine fermentation. Two open reading frames (orfB and orfC) were identified in the upstream region of the hsp18 gene, encoding the small heat-shock protein Lo18. Expression of these genes in conditions of acid stress was studied in Escherichia coli. Methods and Results: Sequence analysis showed that orfB encodes a putative transcriptional regulator of the LysR family. The protein encoded by orfC shares homologies with multi-drug resistance systems. Heterologous expression of orfB, orfC and hsp18 genes in Escherichia coli significantly enhanced the viability of the host strain under acidic conditions. Conclusions: It was demonstrated tha…
Chemosensory Receptors in the Larval Maxilla of Papilio hospiton
2022
Among the butterflies of the genus Papilio (Lepidoptera: Papilionidae), Papilio hospiton (Géné) has a geographical distribution limited to the Mediterranean islands of Sardinia (Italy) and Corsica (France). This is mainly due to the host range that includes only a few plant species of Apiaceae and Rutaceae growing on these islands. In a previous electrophysiological investigation conducted on the maxillary gustatory system of larvae of P. hospiton and its closely phylogenetically related species Papilio machaon, a significantly higher spike activity was shown for the gustatory neurons of lateral and medial styloconic sensilla in P. hospiton when bitter compounds were tested. This effect was…
Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor
2012
Facteur d'impact (5 ans) : 1,617Notoriété à 2 ans : Acceptable (biochem.res.methods); The sweet taste receptor is a heterodimeric receptor composed of the T1R2 and T1R3 subunits, while T1R1 and T1R3 assemble to form the umami taste receptor. T1R receptors belong to the family of class C G-protein coupled receptors (GPCRs). In addition to a transmembrane heptahelical domain, class C GPCRs have a large extracellular N-terminal domain (NTD), which is the primary ligand-binding site. The T1R2 and T1R1 subunits have been shown to be responsible for ligand binding, via their NTDs. However, little is known about the contribution of T1R3-NTD to receptor functions. To enable biophysical characteriza…
Crystallization and preliminary X-ray analysis of strictosidine synthase and its complex with the substrate tryptamine
2005
Strictosidine synthase (STR1) is a central enzyme that participates in the biosynthesis of almost all plant monoterpenoid indole alkaloids. After heterologous expression in Escherichia coli, crystals of STR1 and its substrate complex with tryptamine were obtained by the hanging-drop technique at 302–304 K with potassium sodium tartrate tetrahydrate as precipitant. All crystals belong to space group R3. The native STR1 crystals diffract to 2.95 Å and have unit-cell parameters a = b = 150.3, c = 122.4 Å. The tryptamine complex crystals diffract to 2.38 Å, with unit-cell parameters a = b = 147.3, c = 122.3 Å.
An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities.
2015
Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in Ih activation curve, and an altered responsiveness of Ih to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal …